EFFECT OF AN ENERGY-RESTRICTED

MEDITERRANEAN DIET, PHYSICAL ACTIVITY AND

BEHAVIORAL TREATMENT ON THE PRIMARY
PREVENTION OF CARDIOVASCULAR DISEASE

THE PREDIMED-PLUS TRIAL

RESEARCH PLAN

January 2014

1
 Table of contents

Page

Amendments to the protocol after January 2014 3

2014 PROTOCOL

Abstract 4

Background 6

Hypothesis 11

Objectives 11

Methodology 13

Statistical Analysis Plan 50

Strengths and limitations 53

Committees and governance 54

Sources of funding and administrative issues 55

References 57

Annex I (substudies) 71

Annex II (participating centers) 72

2
AMENDMENTS TO THE PROTOCOL AFTER JANUARY 2014

1. In January 2014 the Steering Committee following the advice of the Data Safety and
Monitoring Board decided to amend the Protocol and omit the 1.5 kg weight loss
criterion to be achieved during the run-in period. Such change only affected the first
70 participants who were eligible and randomized in 2 vanguard centers.
2. September-July 2014. Initially, we decided to provide 500 g per month at no cost of
nuts to each participant during the intervention in order to reinforce adherence to the
Mediterranean diet in both arms of the trial. However, due to lack of economic
resources, only participants included in the pilot study recruited in 2 vanguard centers
up to July 2014 received 250 g of free nuts (125g of pistachios and 125g of almonds
per month) along with the recommendation to consume a total monthly amount of
500g. Subsequently, all trial participants received during the follow-up 125g of
almonds per month out of the total recommended amount of 500g.
3. Four coordinated grants to fund the trial were received from the Instituto de Salud
Carlos III (Madrid) for the periods 2014-2016 and 2017-2019 (Coordinator J. Salas-
Salvado) and for the periods 2015-2017 and 2018-2020 (Coordinator J. Vidal).
4. November 2014. In order to increase the possibility of meeting target numbers of
recruitees by December 2017, in November 2014 the Steering Committee accepted
the inclusion of three additional recruiting centers (see below).

21 Jesús Vioque López vioque@umh.es Universidad Miguel Hernández, Sant

Joan d'Alacant, Alicante
22 Miguel Delgado mdelgado@ujaen.es Universidad de Jaén, Jaén
Rodríguez
23 Vicente Martin vicente.martin@unileon.es Universidad de León, León

5. December 2016. Dr. Francisco Tinahones (Hospital de Málaga) was incorporated as

the seventh member of the Steering Committee.
6. February 2018. Dr. Julia Wärnberg (jwarnberg@uma.es, School of Health Sciences)
replaced the Principal Investigator of the recruitment center at Malaga University
(Preventive Medicine, Medical School), Prof. Enrique Gómez-Gracia, who continued
in the trial as Principal Investigator of a new Support Group (A8).

A8 Enrique Gómez- egomezgracia@gmail.com Preventive Medicine, Medical

Gracia School, University of Malaga

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ABSTRACT
In the 21st century we are witnessing an alarming increase in overweight and
obesity. In Spain, over 60% of adults are overweight or obese and the prevalence of
adult abdominal obesity exceeds 35% (Gutiérrez-Fisac et al., 2012). The increase in
morbid obesity is especially worrying (Basterra-Gortari et al., 2011) because the
medium-to-long-term consequences for the risk of cardiovascular disease (CVD) and
other causes of death or illness can be catastrophic.
Observational studies have shown that all-cause mortality increases progressively
in parallel with adiposity and that the risk of cardiovascular mortality is especially high
(Berrington de González et al., 2010). However, a meta-analysis published in early 2013
(Flegal, 2013) raised considerable controversy by much downgrading the consequences
of overweight and moderate obesity. The results of this meta-analysis could be explained
by the existence of biases, such as unusual definitions for categories of overweight,
tobacco as a confounding factor, inverse causality, and the exclusion of relevant studies
(Tobias and Hu, 2013). Nevertheless, the controversy persists and will probably do so
well into the next few decades. Doubts such as these will only be solved by randomized
intervention trials (Hernán and Taubman, 2008). Thus, clinical studies are needed to
determine whether intentional weight loss reduces cardiovascular mortality and CVD
incidence.
In the PREDIMED-PLUS trial we will evaluate the safety and effectiveness of a
multifaceted intervention program for alleviating excessive cardiovascular morbidity and
mortality in overweight and obese individuals. The study’s aim is to determine the effect
on CVD morbidity and mortality of an intensive weight loss intervention program based
on an energy-restricted traditional Mediterranean diet, increased physical activity and
behavioral therapy in comparison with an intervention based on standard dietary advice
(energy-unrestricted Mediterranean diet) and traditional health care for CVD prevention.
We hypothesize that an intensive lifestyle intervention program aimed at weight loss and
based on the traditional Mediterranean diet is a sustainable long-term approach for
achieving weight loss in overweight and obese adults and that the lifestyle changes
achieved will have a beneficial effect on cardiovascular morbidity and mortality (Estruch
et al., 2013; Shai et al., 2008). This study may provide a useful tool for tackling excess
morbidity and mortality associated with overweight and obesity.

Objectives
The PREDIMED-PLUS trial will evaluate the effect on primary CVD prevention of an
intensive intervention program comprising a 17-item energy-restricted Mediterranean

4
diet with specific weight-loss goals, physical activity promotion and behavioral support
(intervention group) in comparison to a control group using Mediterranen diet
recommendations (comprising a 14-item energy-unrestricted Mediterranean diet) but
without advice to increase physical activity or reduce energy intake (Schröder et al.,
2011) (control group). The diet that will be assigned to the control group has previously
been shown to be effective for CVD prevention in the PREDIMED trial (Estruch et al.,
2013). The primary end-point will be a composite of hard clinical cardiovascular events
(myocardial infarction, stroke or cardiovascular death) as defined in the PREDIMED trial
(Martinez-Gonzalez et al., 2012).

The main objective of PREDIMED-PLUS is to evaluate, in comparison with a control

group given non-intensive, energy-unrestricted dietary advice (also Mediterranean-
type), the effect of an intensive lifestyle intervention comprising an energy-restricted
Mediterranean diet, increased physical activity and behavioral support on:

1. The incidence of cardiovascular events (non-fatal myocardial infarction, non-fatal

stroke, or cardiovascular death).

2. Weight loss and long-term maintenance of weight-loss.

The secondary objectives are to determine whether an intensive weight-loss-

oriented lifestyle intervention program has a beneficial effect to reduce waist
circumference and the following overweight- and obesity-related conditions: acute
coronary syndromes with or without coronary revascularization, heart failure, atrial
fibrillation, peripheral artery disease, venous thrombosis, type-2 diabetes mellitus and its
complications, overall incidence of cancer, specific cancers in main cancer sites (breast,
colorectal, prostate, lung and stomach), osteoporotic fractures, gallstone disease,
symptomatic gout, neurodegenerative disorders (dementia and Parkinson’s disease),
unipolar depression and eating behavior disorders. We will also determine the effect of
the intervention on the following intermediate markers: nutrient intake and overall dietary
pattern, systolic and diastolic blood pressure, blood lipids, fasting glucose level, kidney
function, uric acid level, the percentage of individuals requiring anti-hypertensive, anti-
diabetic or lipid-lowering medication, C-reactive protein levels, hemoglobin A1C levels,
liver function, ECG traits, cognitive function, quality of life, and psychopathological
symptoms.

5
Methodology
We are conducting a randomized, multicenter field trial aimed at the primary
prevention of CVD in overweight or obese adults with metabolic syndrome using an
intensive intervention program based on an energy-restricted Mediterranean diet,
increased physical activity, and behavioral support and a control group given advice on
an ad libitum Mediterranean diet for the prevention of cardiovascular morbidity and
mortality in accordance with the PREDIMED trial. We stress to participants the
importance of attending medical visits and provide them with general written
recommendations on lifestyle for the management of the metabolic syndrome. This new
trial, the planning and design of which is outlined in this paper, is called PREDIMED-
PLUS.
Involved in this new multicenter trial are 20 recruiting centers that aim to recruit a
total of 6,000 participants, of whom 3,000 will be assigned to the intensive intervention
group and 3,000 to the control group. Recruitment takes place between 2013 and 2017.
The intervention will last at least 6 years and the median follow-up time for the clinical
endpoints is expected to be 8 years. The results of the trial, including anthropometric
changes and impact on major obesity-related disorders, are expected to be highly
applicable to public healthcare since they will provide a better prognosis for overweight
and obese adults. The results are also expected to be highly efficient since they should
provide a non-pharmacological approach to the prevention of the main cause of mortality
and one of the leading causes of loss of disability-adjusted life years (Lozano et al.,
2012).

6
BACKGROUND
The global overweight and obesity epidemic is increasing at an alarming rate.
Now a global public health crisis, it affects over 60% of the adult population. Between
1980 and 2008, worldwide obesity prevalence almost doubled (Finucane et al., 2011;
Malik et al., 2013). In Spain, the prevalence of adult abdominal obesity is over 35%
and over 60% of the adult population is overweight or obese (Gutiérrez-Fisac et al.,
2012). Moreover, there is a particularly worrying increase in morbid obesity (Basterra-
Gortari et al., 2011). The medium-to-long-term consequences of obesity on the risk of
CVD and death are devastating and have the capacity to both render the health
system unsustainable and curtail economic growth. Urgent priority must be given to
finding solutions based on the best scientific evidence available.
The link between obesity and mortality appears to diminish with age. However, if this
were true, it would be difficult to recommend weight loss for older populations. This notion
has been challenged by observational epidemiological studies that, after generational
and cohort confusion are adequately controlled, suggest the opposite, i.e., that with
advancing age the link between obesity and mortality becomes even stronger (Masters,
2013).
A meta-analysis of observational studies published in 2013 (Flegal, 2013) raised
controversy by considerably downgrading the consequences of overweight and
moderate obesity. However, as Tobias and Hu (2013) have argued, the conclusions of
Flegal’s meta-analysis can probably be explained by the existence of biases, such as
unusual definitions for categories of overweight, tobacco as a confounding factor, inverse
causality, and the exclusion of relevant studies. This issue will only be solved by
randomized clinical trials (Hernán and Taubman, 2008). However, the controversy is
likely to continue well into the next few decades until the results of clinical intervention
studies are available that overcome the limitations and inherent bias of the observational
designs that have evaluated the link between overweight or obesity and the incidence of
serious clinical events or mortality in initially healthy individuals. Observational studies
with a better control of bias have found that all-cause mortality increases progressively
as adiposity outside the normal weight range—measured by body mass index (BMI,
defined as weight in kilograms divided by the square of height in meters)—increases,
and that this risk is especially high for cardiovascular mortality (Berrington de
González et al., 2010). An increase in body weight is associated not only with higher
mortality but also with greater morbidity due to CVD (Ni Mhurchu et al., 2004; Song et
al., 2004; Flint et al., 2010); greater risk of developing some types of cancer, diabetes
and depression (Luppino et al., 2010); and poorer cognitive function (Gunstad et al.,

7
2010). Large-scale randomized studies with robust designs are needed to obtain best-
quality evidence.
Expert panels set up by the National Institutes of Health and the World Health
Organization recommend that overweight and obese adults with comorbid conditions
should lose 10% of their initial weight and that a lifestyle intervention program should be
the primary treatment (National Institute of Health, 1998; World Health Organization,
1998). Moreover, according to the American Dietetic Association, a weight-loss-oriented
intensive lifestyle intervention program should include an energy-restricted diet, physical
activity and behavioral therapy. The only randomized trial that has addressed the long-
term effect of an intensive weight-loss lifestyle program in obese adults on CVD and
mortality is the Look AHEAD study (Ryan, 2003). This trial, which comprised 5,145
participants (Rejeski et al., 2012), ended prematurely in October 2012 due to lack of
efficiency (Look AHEAD Research Group, 2013). The trial included only diabetic subjects
and used a low-fat diet (<30% of total energy intake with <10% from saturated fat). To
some extent this is opposite to the Mediterranean diet used in the PREDIMED trial
(Zazpe et al., 2008; Martínez-González et al, 2012; Estruch et al., 2013), which is rich in
vegetable fats such as healthful virgin olive oil and nuts. In recent years, scientific
associations have recommended low-fat diets that contribute less than 30% of energy in
the form of fat as the most suitable way to promote general health and weight loss. The
high energy density and high palatability of high-fat foods are feared to produce
potentially adverse effects on body weight and cardiovascular health (National Institute
of Health, 2000). However, the discontinuation of the Look AHEAD trial due to futility, the
inefficiency of the Women’s Health Initiative Dietary Modification Trial (Look Ahead
Research Group, 2013; Howard, 2006), and the favorable results of the PREDIMED
(Estruch et al., 2013) and DIRECT studies (Shai et al., 2008) provide powerful arguments
against approaches based on low-fat diets.
Although diets that recommend complex carbohydrates, a reduction in fat intake and
energy restriction to produce weight loss are generally accepted, there is no clear
evidence that dietary fat is associated with a greater increase in weight (Willett, 2001;
Nordmann et al., 2006; Larsen et al., 2010; Hu et al., 2012; Bueno et al., 2013). One
dietary paradigm that is different from the low-fat diet and that can be more useful for
developing and implementing programs aimed at achieving prolonged weight loss and
improving the metabolic alterations associated with overweight and obesity is the
traditional Mediterranean diet. This dietary pattern is rich in fat from vegetable sources
(virgin olive oil and nuts) and includes an abundance of minimally processed plant foods
(vegetables, fruits, whole grains and legumes), low consumption of meat (especially red

8
and processed meats), moderate consumption of fish and wine (which is usually
consumed with meals) and frugal meals. The high fat contents of the traditional
Mediterranean diet make the diet more palatable and therefore more acceptable and
easily sustainable in the long term.
In its 2010 edition, the Dietary Guidelines for Americans recognized the traditional
Mediterranean diet, together with the DASH diet, as a healthy diet for the prevention of
CVD, although when the recommendation was made, randomized clinical trials with
regard to the primary prevention of major clinical events as the main outcome had not
yet been conducted. This was confirmed by the results of the PREDIMED trial on the
primary prevention of cardiovascular disease conducted in Spain between 2003 and
2010 (Estruch et al., 2013).
With regard to the weight-loss properties of the traditional Mediterranean diet, in a
meta-analysis of randomized trials, allocation to a Mediterranean diet in comparison with
control diets showed a small but significant effect on body weight reduction (mean
differences: -1.75 kg, CI 95%: -2.86 a -0.64 kg). This effect was doubled when the
Mediterranean diet was energy-restricted (Esposito et al., 2011). Another meta-analysis
of observational studies (Sofi et al., 2010) found that greater adherence to the
Mediterranean diet was associated with significant reductions in total mortality,
cardiovascular mortality, mortality due to cancer, the incidence of non-fatal
cardiovascular events, and the risk of neurodegenerative illnesses. A subsequent update
of the meta-analysis of the Mediterranean diet and CVD reported a 13% relative
reduction in risk for every two-point increase in adherence to the Mediterranean diet
(scale 0–9) after identifying and treating sources of heterogeneity (Martínez-Gonzalez
and Bes-Rastrollo, 2014).
The PREDIMED (PREvención con DIeta MEDiterránea) trial, which included
7,447 participants over an average of five years, was the largest nutritional intervention
trial ever conducted in Europe. PREDIMED showed that, in comparison with advice on
a low-fat diet, a high-fat Mediterranean diet supplemented with extra-virgin olive oil or
mixed nuts implemented in a setting of primary cardiovascular prevention resulted in a
30% reduction in CVD events after intervention for a median of 4.8 years (Estruch et
al., 2013). PREDIMED is recognized worldwide as a landmark study that marks a turning
point in the prevention of chronic diseases. The effective reduction in cardiovascular
events when the Mediterranean diet was used in a randomized trial provides the best-
possible scientific evidence for preventing CVD, the main cause of death in the world.
We should also point out that the PREDIMED diets were ad libitum, increased physical
activity was not promoted, and no counsel to lose weight was given.

9
 It has been postulated that the link between adherence to the traditional
Mediterranean diet and the risk of CVD can be mediated by several mechanisms,
including a reduction in low degree inflammation (Chrysohoou et al., 2004; Esposito et
al., 2004; Mena et al., 2009; Camargo et al., 2011; Urpi-Sarda et al., 2012; Meneses et
al., 2012), higher levels of adiponectin (Detopoulou et al., 2010; Razquin et al., 2010),
lower coagulability (Chrysohoou et al., 2004; Pérez-Jiménez et al., 2006; Pérez-Jiménez
et al., 2002), improved endothelial function (Esposito et al., 2004; Ruano et al., 2005;
Fuentes et al., 2008), lower oxidative stress (Dai et al., 2008; Chrysohoou et al., 2011;
Razquin et al., 2009), a lower concentration of atherogenic lipoproteins (Jones et al.,
2012), lower levels of oxidized LDL particles (Fito et al., 2007), and a lower uptake of
oxidized LDL by macrophages (Moreno et al., 2008). Moreover, the two foods
supplemented in PREDIMED (extra-virgin olive oil and nuts) also have beneficial
biological properties. Extra-virgin olive oil has a healthy fatty acid profile and contains
numerous bioactive phenolic compounds (Pérez-Jiménez et al., 2006; Covas et al.,
2009; López-Miranda et al., 2010). The phenolic compounds of olive oil have anti-
inflammatory properties (Fito et al., 2008), beneficially impact he lipid profile (Benkhalti
et al., 2002; Covas et al., 2006), improve oxidative stress markers (Covas et al., 2006),
have a platelet antiagregant effect (de Roos et al., 2011; Fito et al., 2008), and stimulate
mitochondrial biogenesis (Zhu et al., 2010). Nuts also have a healthy fatty acid profile,
based on mono- and polyunsaturated fatty acids, and contain minerals, vitamins and
other antioxidant bioactives, essential amino acids, fiber, and phytosterols (Ros, 2009).
The consumption of nuts has been associated with lower levels of total cholesterol, LDL
and non-HDL cholesterol, and apolipoprotein B-100 (Li et al., 2009; Sabaté et al., 2010),
and lower inflammation (Jiang et al., 2006). Nuts also have an antioxidant effect, benefit
heart rate and improve platelet aggregation and endothelial function (Ros, 2009;
Defilippis et al., 2010). All of these mechanisms explain the antiatherogenic effect of a
Mediterranean diet that is rich in nuts and extra-virgin olive oil. In fact, in the PREDIMED
trial a strong protective effect against peripheral artery disease was observed (Ruiz-
Canela et al., 2014). In a sub-study of the PREDIMED trial we also observed that both a
Mediterranean diet enriched with nuts and a Mediterranean diet enriched with olive oil
reduced the incidence of type-2 diabetes by 48% (Salas-Salvadó et al., 2011). When we
analyzed this association among all study’s participants, we also found that the
Mediterranean diet had a significant protective effect against diabetes (Salas- Salvadó
et al., 2014).
Though PREDIMED study, was not a weight loss trial, the provision of abundant fat-
rich foods from natural vegetable origin (extra-virgin olive oil and nuts) did not conduct

10
to weight gain. There is still insufficient experimental evidence to support the
hypothesis that intentional weight loss via a healthy diet and favorable lifestyle changes
reduces mortality or the incidence of CVD in the long term. Specifically, the impact of
weight loss on the risk of CVD within the framework of a Mediterranean dietary pattern
has not yet been tested in a sufficiently large randomized clinical trial (Malik and Hu,
2007). In light of the obesity epidemic, we propose to conduct a new trial, PREDIMED-
PLUS, which will go beyond the achievements of the PREDIMED trial in order to tackle
more specifically the problems of overweight and obesity. Our proposed strategy has
positive effects for weight loss (based on the loss of fat mass) and long-term weight-loss
maintenance (Shai et al., 2008; Beunza et al., 2010; Romaguera et al., 2010). Even more
interestingly, this research may demonstrate that a multifaceted lifestyle intervention
program (dietary pattern + weight loss + physical activity + behavioral support) can be
an even more effective means of reducing the cardiovascular risk associated with
overweight and obesity than a non-energy-restricted traditional Mediterranean diet. We
expect our contribution via the PREDIMED-PLUS trial to reveal synergies between the
effects of an intensive weight-loss intervention program (with energy restriction,
physical activity and behavioral support) and the beneficial effects of greater adherence
to a high-quality diet (the Mediterranean diet) on the incidence of CVD.
Blood and urine samples will be collected and stored at the beginning and
throughout the trial. Later analyses of molecular/biochemical biomarkers within the
framework of genetic, epigenetic, transcriptomic, metabolomic and proteomic studies
might help to determine the benefits of the intervention and the underlying mechanisms.

HYPOTHESIS
An intensive lifestyle intervention program based on an energy-restricted
traditional Mediterranean diet, increased physical activity and behavioral support is a
sustainable approach that leads to long-term weight loss in overweight and obese adults
with metabolic syndrome in such a way that the changes in lifestyle achieved will have
long-term benefits on the incidence of CVD.

In comparison with a control intervention that provides advice on the Mediterranean

diet but does not restrict calorie intake and does not promote physical activity, an
intensive lifestyle intervention based on an energy-restricted traditional Mediterranean
diet, promotion of physical activity, and behavioral support (Intervention group) in
overweight or obese individuals with metabolic syndrome will:

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1. Reduce the risk of cardiovascular events;
2. Achieve a greater reduction in body weight and lead to better long-term weight-loss
maintenance;

OBJECTIVES
Our long-term objective is to provide effective treatment for reducing excessive
cardiovascular morbidity and mortality in overweight or obese adults, irrespective of
whether the participants are diabetic at the beginning of the study. To achieve this, we
will compare the effects on rates of cardiovascular disease of an intensive lifestyle and
weight loss intervention program based on the traditional Mediterranean diet and
including increased physical activity, energy restriction and behavioral support
(intervention group) with that of a non-intensive intervention program that provides both
education on the traditional Mediterranean diet for the prevention of CVD in
accordance with the principles outlined in the PREDIMED trial and usual care by
primary healthcare professionals (control group). The importance of attending visits to
healthcare professionals will be stressed and general recommendations on management
of the metabolic syndrome will be provided.

Main specific objectives

To evaluate the effect of an intensive weight-loss-oriented lifestyle intervention program
based on a traditional Mediterranean diet with energy restriction, increased
physical activity and behavioral therapy on:
1. The incidence of CVD (non-fatal myocardial infarction, non-fatal stroke and
cardiovascular death);
2. Weight loss and long-term weight-loss maintenance;

Secondary specific objectives

This intensive intervention program is likely to result in reduction of waist
circumference and acute coronary syndromes, coronary revascularization, total
mortality, heart failure, peripheral artery disease, venous thrombosis, atrial fibrillation,
type-2 diabetes and its complications, total cancer, cancer in main cancer sites (breast,
prostate, colorectal, lung and stomach), gallstone diseases, symptomatic gout,
neurodegenerative disorders (dementia and Parkinson’s disease), unipolar depression,
osteoporotic fractures, and eating behavior disorders.
We will also address the effect of the intervention on the following intermediate
outcomes: nutrient intake and overall dietary pattern, systolic and diastolic blood

12
pressure, serum lipid concentrations, fasting glucose, glycated hemoglobin and uric acid,
kidney function, liver function, C-reactive protein, anti-hypertensive, anti-diabetic and
lipid-lowering medication needs, ECG traits, cognitive function, quality of life, and
psychopathological scales.
We will also store plasma, serum, peripheral cells and urine samples to evaluate
other hypotheses in the future, depending on availability of additional funding.

METHODOLOGY
1. Summary
We are conducting a parallel-group, multicenter, randomized, primary prevention
trial in adult men aged 55–75 and adult women aged 60–75 with a BMI ≥27 and <40
kg/m2 who meet at least three criteria for the metabolic syndrome. In the Caucasian
population, cardiovascular risk is considered to be increased if waist circumference is ≥
80 cm in women and ≥ 94 cm in men. In the South American population, the value is the
same for women but for men risk is considered to be increased if waist circumference is
≥ 90 cm (Alberti et al., 2009). Diabetic participants will comprise no more than 25% of
the final sample. This latter component and the use of the traditional Mediterranean
diet will substantially distinguish the PREDIMED-PLUS trial from the Look AHEAD trial
conducted in the United States (Ryan et al., 2003; Gregg et al., 2012; Look AHEAD
Research Group, 2013), which was recently discontinued due to lack of efficiency.
Participants will be divided proportionally at random into two groups: a control group and
an intensive lifestyle intervention group. Intervention will be maintained for 6 years and
average follow-up time for clinical events will be 8 years.
The control group receives usual healthcare from primary care medical
professionals, all the written material, instructions on following the traditional
Mediterranean diet—which was used in the PREDIMED study and has been shown to
have benefits for the prevention of cardiovascular morbidity and mortality—and general
lifestyle recommendations for managing the metabolic syndrome. Every six months, the
control group participants are also invited to participate in group sessions led by the team
of PREDIMED-PLUS dietitians, wherein they receive a free supply of virgin olive oil (6
liters every 6 months) and nuts (3 kg every 6 months) in order to promote the
Mediterranean diet and encourage compliance with the trial.
Participants in the intensive lifestyle intervention group are prescribed a
traditional Mediterranean diet but in this case it is energy-restricted. Dietary
intervention is associated with increased physical activity and behavioral therapy
programs. It has specific weight-loss objectives and includes self-monitoring and

13
frequent follow-up throughout the study. Participants in this group take part in individual
interview sessions and motivational group sessions three times per month during the first
year of the intervention and twice per month thereafter. They are provided free extra-
virgin olive oil (one liter per month) and nuts (500 g per month) . The participants’ degree
of compliance with the intervention is monitored periodically so that the intervention can
be adjusted if necessary.
For the intensive intervention group, the specific weight-loss objectives are to
achieve an average reduction in baseline body weight of over 8% and an average
reduction in waist circumference of over 5% in the first six months and to maintain
these figures over an additional period of seven and a half years. The final objective is
to obtain a between-group average absolute difference in weight loss and waist
circumference reduction of over 5%.
Primary final outcomes include: a) non-fatal myocardial infarction, b) non-fatal
stroke, and c) cardiovascular death. Other primary objective will be weight loss (and
weight-loss maintenance). The trial protocol will be registered at ClinicalTrials.gov
(National Institutes of Health) and comply with the CONSORT guidelines for the
dissemination of results (Moher et al., 2001).

2. Research team
The trial will comprise 6,000 participants, one-half of whom will be assigned to the
intensive intervention group and one-half to the control group. Recruitment of participants
began in 2013 and will end in 2017. The 2 vanguard centers were Navarra-Epidemiology
(starting the recruitment in September 2013 and the randomization in October 2013) and
Reus (starting the recruitment in November 2013, and the randomization in January
2014). Training of dietitians for the other recruiting centers took place in December 2013.
We assume that we will reach our recruitment objectives with 20 centers each recruiting
an average of 300 participants.
To launch the trial, we have set up a team of leading researchers with experience in
diet and lifestyle interventions and productive and well-documented collaborative

The decision to administer 500 g of mixed nuts per month was based on the results of epidemiological and
clinical studies. For example, the SUN (Seguimiento Universidad de Navarra) study found that individuals
who consumed nuts two or three times per week (400 g/month) had a significantly lower risk of weight gain
and metabolic syndrome than those who rarely ate them or never did (Fernández-Montero et al., 2012;
Bes-Rastrollo et al., 2007). A Mediterranean-type diet with moderate fat intake containing 25 g per day
(750 g/month) of peanuts or other types of nuts was found to be associated with better adherence to
intervention and greater weight loss than a low-fat diet (McManus et al., 2001). The decision was also
based on associations observed between baseline nut consumption and mortality in the PREDIMED trial
(Guasch-Ferre et al., 2013) and in American cohorts of nurses and healthcare professionals (Bao et al.,
2013).

14
research careers in nutrition, evaluation of physical activity, internal medicine,
cardiology, endocrinology, primary health care, epidemiology, and basic sciences.
The prior experience gained by the 11 PREDIMED recruiting centers is one of our
most valuable assets for the PREDIMED-PLUS trial. Also, by incorporating other centers
of scientific excellence with proven experience in nutritional intervention clinical trials
(some of which also belong to the background research network of the PREDIMED trial,
CIBEROBN), the correct development of the PREDIMED-PLUS trial will be guaranteed
and feasibility will be improved.
At the same time, subprojects will be devised so that all participating groups can
develop their own specific nutritional research activities. This will also serve to further
interest in the project and enhance scientific output.

3. Preliminary studies: the PREDIMED trial

In this section we present a summary of the methodology and key findings of the
PREDIMED trial (Estruch et al., 2006; Zazpe et al., 2008; Martínez-González et al., 2012;
Estruch et al., 2013), which was conducted in the context of CIBEROBN and the
PREDIMED network (RD06/0045). PREDIMED was a multicenter, parallel group, trial
with three intervention groups (see www.predimed.es). In 2006, the results of a pilot
study were published that evaluated the effects at three months of the three interventions
on classical and emergent cardiovascular risk factors in the first 772 participants (Estruch
et al., 2006). The design and methods of the PREDIMED trial have been described
elsewhere (Martínez-González et al., 2012). Participants in the study were men aged 55-
80 and women aged 60-80 without CVD at the beginning of the study but with a high risk
of CVD due to the presentation of type 2 diabetes or at least three of the following six
cardiovascular risk factors: smoking, high blood pressure, high LDL cholesterol level, low
HDL cholesterol level, overweight or obesity, and family history of early coronary heart
disease.
Candidates for the trial were recruited at primary care health centers. Eighty-nine
per cent of those invited to participate agreed to do so and signed the corresponding
informed consent form. The final sample size for the trial was 7,447 participants. The
protocol was approved by the ethics research committees of all study centers and
registered at the Clinical Trials Register in London (ISRCTN35739639).
The participants were randomly assigned in a 1:1:1 ratio to one of the following three
dietary intervention groups: 1) an energy-unrestricted Mediterranean diet
supplemented with extra-virgin olive oil; 2) an energy-unrestricted Mediterranean diet

15
supplemented with nuts; or 3) an energy-unrestricted control diet with advice on how to
follow a low-fat diet.
At the beginning of the study and quarterly thereafter, dietitians conducted individual
and group dietary-training sessions (separately for each group) with a maximum of 20
participants per group (Zazpe et al., 2008). At each session of the Mediterranean diet
groups, a 14-item questionnaire (Martínez-González et al., 2004; Schroeder et al., 2011)
was used to assess participants’ adherence to the Mediterranean diet. At each session
of the control group, a 9-item questionnaire was used to assess participants’ adherence
to the control diet. In this way the diets could be personalized and appropriate dietary
changes could be negotiated individually.
Participants in the two Mediterranean-diet groups received either extra-virgin
olive oil (1 liter per week for the participant and his or her family) or 30 g of mixed nuts
per day (15 g of walnuts, 7.5 g of hazelnuts, and 7.5 g of almonds) at no cost and in
accordance with their randomly chosen group, while those in the control group received
small non-food gifts throughout the trial. At no point during the intervention was calorie
restriction advised or increased physical activity encouraged.
Information on the primary end points (cardiovascular death, non-fatal stroke, or
acute non-fatal myocardial infarction) was obtained through continuous contact with the
participants, contact with primary healthcare physicians, ad hoc annual reviews of
medical records conducted by a medical team at each center, and annual consultation
of the National Death Index. The data were analyzed on an intention-to-treat basis.
Participants were followed for a median of 4.8 years.
Participants in the two Mediterranean diet groups increased their adherence to the
diet, as reflected in an average increase of 2 points on the 14-point dietary-screening
questionnaire throughout the duration of the study. These participants also had higher
average scores than those in the control group on all items except red and processed
meats and sugary soda drinks, which were discouraged for all three intervention groups.
After a median follow-up of 4.8 years, 288 participants suffered a major
cardiovascular event. In comparison with the control group, the multivariable-adjusted
hazard ratios were 0.70 (95% confidence interval [CI], 0.54 to 0.92) for the group
assigned to the Mediterranean diet with extra-virgin olive oil and 0.72 (95% CI, 0.54 to
0.96) for the group assigned to the Mediterranean diet with nuts.

16
FIGURE 1. Cumulative incidence of major cardiovascular events (cardiovascular
death, non-fatal myocardial infarction, and non-fatal stroke) by randomly assigned
group (n=7,447).

4. Proposed trial (PREDIMED-PLUS) and eligibility criteria

Candidates for the PREDIMED-PLUS trial are adults aged 55-75 for men and 60-75
for women with a body mass index ≥27 and <40 kg/m2 who meet at least three criteria
for the metabolic syndrome (Alberti et al., 2009). These criteria must be taken into
consideration in view of recent evidence of the beneficial role of the Mediterranean diet
on the metabolic syndrome (Kastorini et al., 2011; Salas-Salvadó et al., 2008), insulin
resistance and diabetes (Salas-Salvadó, 2014), especially when accompanied by a
program of physical activity for endurance (Fernández et al., 2012). We will try to ensure
that 50% of the study population is made of women and that diabetic participants do not
exceed 25% of the total cohort. Individuals who participated in the PREDIMED trial will
not be eligible to participate in PREDIMED-PLUS.

4.1. Exclusion criteria:

• Illiteracy or inability/unwillingness to give written informed consent or communicate with
study staff.
• Institutionalization (the participant is a permanent or long-stay resident in a care home).

17
• Documented history of previous CVD, including: angina; myocardial infarction; coronary
revascularization procedures; stroke (ischemic or hemorrhagic, including transient
ischemic attacks); symptomatic peripheral artery disease that required surgery or was
diagnosed with vascular imaging techniques; ventricular arrhythmia; uncontrolled
atrial fibrillation; congestive heart failure (New York Heart Association Class III or IV);
hypertrophic myocardiopathy; and history of aortic aneurism >=5.5 cm in diameter or
aortic aneurism surgery.
• Active malignant cancer or history of malignancy within the last 5 years (except non-
melanoma skin cancer).
• Inability to follow the recommended diet (for religious reasons, swallowing disorders,
etc.) or to carry out physical activity.
• A low predicted likelihood to change dietary habits according to the Prochaska and
DiClemente Stages of Change Model (Nigg et al., 1999).
• Inability to follow the scheduled intervention visits (institutionalization, lack of autonomy,
inability to walk, lack of stable address, travel plans, etc.).
• Inclusion in another program that provides advice on weight loss (> 5 kg) in the six
months before the selection visit.
• History of surgical procedures for weight loss or intention to undergo bariatric surgery
in the next 12 months.
• History of small or large bowel resection.
• History of inflammatory bowel disease.
• Obesity of known endocrine origin (except for treated hypothyroidism).
• Food allergy to any component of the Mediterranean diet.
• Immunodeficiency or HIV-positive status.
• Cirrhosis or liver failure.
• Serious psychiatric disorders: schizophrenia, bipolar disorder, eating disorders, or
depression with hospitalization within the last 6 months.
• Any severe co-morbidity condition with less than 24 months’ life expectancy.
• Alcohol abuse or addiction (or total daily alcohol intake >50 g) or drug abuse within the
past 6 months.
• History of major organ transplantation.
• Concurrent therapy with immunosuppressive drugs or cytotoxic agents.
• Current treatment with systemic corticosteroids.
• Current use of weight loss medication.
• Concurrent participation in another randomized clinical trial.

18
• Patients with an acute infection or inflammation (e.g. pneumonia) will be allowed to
participate in the study 3 months after resolution of their condition.
• Any other condition that may interfere with adherence to the study protocol.

5. Recruitment and retention strategies

Medical doctors from primary care centers associated with the recruiting centers
recruit the participants. The mission of the primary care physicians is to ensure a high
recruitment rate and an almost 100% diligence in the revision of medical records and
collection of clinical information on events during follow-up. As the physicians involved
in the recruitment process will also be responsible for the participants’ medical care, no
potential ethical conflict regarding confidentiality exists when identifying suitable
candidates or reviewing medical records. Participants’ eligibility criteria and demographic
data are collected from the medical records at the primary care centers, which are
entirely computer-based. This is done at a pre-screening evaluation stage before the
potential participant is contacted. Candidates are interviewed briefly by telephone,
informed about the study, and invited to attend a screening visit at the recruiting center.

In this first formal visit (first screening visit), the candidates are explained the
purpose and characteristics of the study and, if they agree to take part, they are asked
to sign a written informed consent. Our experience with the PREDIMED trial showed that
over 95% of eligible candidates approached in this way agreed to participate (Martínez-
González et al., 2012; Estruch et al., 2013). Also, in the PREDIMED trial, which also
included a long-term lifestyle intervention program (median follow-up time of 4.8 years),
overall retention rate was above 90% (Estruch et al., 2013).

6. Informed consent/Ethics Committee

The institutional review boards of all the recruiting centers approved the study
protocol. As described and detailed below, all participants sign informed written consent
forms.

7. Launch of the trial

The proposed calendar was as follows:
• Between August 2013 and January 2014, the dietitians and nursing staff were hired
and trained to deliver the trial’s protocol.
• Between August 2013 and May 2014, eligible candidates began to be called,
interviewed and invited to participate in the trial.

19
• Next, the first evaluation and intervention visits took place with randomly distributed
selected participants.
• The recruitment period will end on December 2016.

7.1. The aim of the initial stage of the trial (telephone calls and interviews) is to evaluate
the willingness of each candidate to participate in the study, comply with the proposed
intervention, and lose weight. In addition, they are thoroughly screened in order to ensure
that the eligibility criteria are met and evaluate the probability that they will:
a) attend the scheduled sessions,
b) complete the protocol’s assessment tools, i.e., the self-monitoring and recording of
lifestyle and food habits and, most importantly,
c) change their dietary habits in accordance with the Stages-of-Change model (Nigg
et al., 1999), as occurred in the PREDIMED trial.

7.2. The run-in period (for evaluation prior to randomization) lasts four weeks. It
comprises an initial screening visit, a phone call at 2 weeks, and a final evaluation visit.
7.2.1. The first screening visit (45-60 min) comprises:
a) administration of a questionnaire on inclusion and exclusion criteria. Candidates
who are deemed eligible to participate in the trial continue to the next stage.
b) explanation of the study, distribution of the study information sheet, and
completion of the informed consent forms (these are essential for inclusion in the
trial). Eligible candidates are asked to sign two informed consent forms: one for
participation and analysis of general variables and one for the collection of DNA for
genetic analyses. All procedures and anticipated time commitment are explained in
detail. Candidates are also told that, if they do not satisfy the eligibility criteria, they
will be excluded from the study. The informed consent form includes a statement
allowing researchers to review the participants’ medical records throughout the trial
at both the primary care centers and reference hospitals in order to ascertain the
occurrence of any events.
c) performance of ECG and recording of height, weight, waist circumference and
blood pressure.
d) distribution of a leaflet containing general recommendations on managing the
metabolic syndrome.
e) Distribution of a 3-day food record questionnaire (2 working days and 1 weekend
day), a leisure-time physical activity questionnaire, and a self-measurement chart in
which participants self-record their weight, waist circumference and hip circumference

20
 (participants are given a tape measure). Dietitians give instructions on how and when
to complete the food record and physical activity questionnaires and how to record
their weight, waist and hip measurements (once a week during the trial).
f) Distribution of the clinical psychopathological questionnaires (Beck Depression
Inventory (BDI-II), multidimensional scale of weight locus of control, eating disorders
diagnostic criteria, and quality of life scale (SF-36)) to be completed at home (see
below).
Participants are asked to return their completed questionnaires at the third screening
visit (see below).
7.2.2. Second screening visit. After 2 weeks, participants receive a telephone call to
assess their change in weight and remind them to bring to the next screening visit
their completed food record, and physical activity questionnaires, self-measurement
chart, psychopathological questionnaires, and quality of life scale.
7.2.3. Third screening visit. This evaluation visit on completion of the four-week run-in
period (30 min) includes:
a) Collection by the dietitian of the participants’ food record and physical activity
questionnaires, self-measurement charts, psychopathological questionnaires, and
quality of life scales.
b) Measurement by the dietitian of the participants’ weight and hip circumferences.
c) Administration and completion of a 143-item food-frequency questionnaire and 5
cognitive-neuropsychological tests, which, unlike the clinical psychopathological
questionnaires, must be completed in the presence of PREDIMED-PLUS personnel.
These 5 tests are: the Mini-Mental State examination, the phonological verbal fluency
test, the reverse digits test, the trail making test, and the clock test (see below).
d) Explanation to participants that they will be informed by telephone if they have been
selected to participate in the trial.
e) Explanation of night-time fasting for in situ extraction of blood sample and first
morning urine sample, and of basal evaluation immediately after randomization for
candidates who are chosen to participate in the trial.

Only candidates who satisfy the following four criteria are selected and randomly
assigned to one of the two intervention groups:
1) Full attendance at the two previous sessions, at the scheduled times and having
answered the telephone call;

21
2) Correct completion of the clinical psychopathological questionnaires (Beck
Depression Inventory (BDI-II), multidimensional scale of weight locus of control, eating
disorders diagnostic criteria, and quality of life scale (SF-36);
3) Correct completion of the food record and physical activity questionnaires;
4) Correct self-recording of at least three weight measurements and three waist-
circumference measurements.
5) Loss of >1.5 kg during the run-in period.

The lag time between completion of the run-in phase and the beginning of the
intervention ranges from one week to one month.
Exceptionally, depending on the availability of the candidate to attend the Run-in S3 visit,
and considering that the 4 mentioned criteria are met, the candidates can be randomized
to one of the two intervention groups after completing Run-in S2 visit face-to-face.

8. Initial screening, follow-up visits, and evaluations

Table 1 shows the main data collection measurements and activities by visit.

22
TABLE 1. The following data are collected per visit in the PREDIMED-PLUS trial.
RUN-IN PERIOD
Baselin Y-8
S1 S2 S3 e
M-6 Y-1 Y-2 Y-3 Y-4 Y-5 Y-6 Y-7
1. ELIGIBILITY QUESTIONNAIRE X
2. 3-DAY FOOD REGISTER e c
3. ANTHROPOMETRIC MEASUREMENTS* X X X X X X X X X X X X
4. GENERAL QUESTIONNAIRE X
5. 143-ITEM FFQ X X X X X X X X X X
6. MEDITERRANEAN DIET QUESTIONNAIRE (17/14-Items)** X X X X X X X X X X
7. PHYSICAL ACTIVITY QUESTIONNAIRE‡ e† c† X X X X X X X X X X
8. CHAIR TEST (Physical activity evaluation) X X X X X X X X X X
9. ACCELEROMETERS e X X X X X X X X X X
10. FOLLOW-UP QUESTIONNAIRE X X X X X X X X X
11. ELECTROCARDIOGRAM X X X X X X X X X X
12. BLOOD PRESSURE MEASUREMENT X X X X X X X X X X X X
13. BLOOD SAMPLE COLLECTION X X X X X X X
14. MORNING SPOT URINE COLLECTION X X X X X X X
15. NAIL COLLECTION X X X X X X
16. COGNITIVE-NEUROPSYCHOLOGICAL TESTS≠ X X X X X
17. PSYCHOPATHOLOGICAL QUESTIONNAIRES€ e X X X X X X X X X
18. QUALITY OF LIFE QUESTIONNAIRES≈ e X X X X X
S: Screening visit; FFQ: Food-frequency questionnaire; M: Month; e: Delivery ; c: Collection.
* Anthropometric measurements include: weight, height, waist circumference and hip circumference.
‡
Short version of the Minnesota leisure time physical activity questionnaire; PAR-Q, RAPA (RAPA1 and RAPA2) questionnaires; and the NHS sedentary lifestyle questionnaire
†
Long version of the Minnesota leisure time physical activity questionnaire.
**Short questionnaires on adherence to the Mediterranean Diet. The control group uses the same 14-item questionnaire that was used in the PREDIMED trial (Schroeder et al.,
2011). The intervention group uses the 17-item energy-restricted Mediterranean diet questionnaire (see below).
≠
Mini-Mental Status Examination, clock test, phonological verbal fluency test (animals + P), the reverse series of digits test (WAIS-III), and the trail making test.
€
Beck Depression Inventory (BDI-II), multidimensional scale of weight locus of control, eating disorders diagnostic criteria, and SF-36 quality of life scale.

23
Eligibility: Eligibility for inclusion in the trial is assessed at the beginning of the study.
Selected participants must satisfy all the eligibility criteria. Exclusion criteria are also
verified.
Anthropometric measurements: Weight and waist circumference will be recorded at each
visit, with participants in light clothing and without shoes or accessories, using a high-
quality electronic scale that will be calibrated every 3 months with a unit of known mass.
Height is measured at study entrance with a stadiometer. Waist circumference is
measured midway between the lowest rib and the iliac crest. Hip circumference is
measured at the widest part at the baseline visit and on a yearly basis.
General information: Information on medical history, family history and use of medication
is collected at the baseline visit by means of the general questionnaire, using the same
protocol as in the PREDIMED trial (See supplementary file in Estruch R et al., New Engl
J Med 2013).
Evaluation of food habits and dietary intake: The previously validated 143-item food-
frequency questionnaire is administered at the third screening visit and at each annual
follow-up visit to evaluate the diet of each participant (Fernández-Ballart et al., 2010). In
addition, the 17-item energy-restricted Mediterranean diet questionnaire (see below) is
completed at each visit. This questionnaire, which includes several changes with respect
to a previously validated tool (Schroder et al., 2011), is used both to assess participants’
compliance with the intervention and to guide the individual motivational interviews
during follow-up. The control group, on the other hand, is administered the same 14-item
questionnaire that was used for the PREDIMED trial (Schröder et al., 2011).
Physical activity: Except for the first screening visit (when participants complete the long
Minnesota leisure time physical activity questionnaire), at the beginning of the study,
after 6 months, and during the follow-up visits, participants will complete a short version
of a previously validated physical activity questionnaire (Elosua et al., 1994; Elosua et
al., 2000). Also at these latter visits (baseline, after 6 months, and during the follow-up
visits), participants will perform the chair test (30 seconds) in order to evaluate their
physical fitness and complete the following questionnaires: the PAR-Q (Physical Activity
Readiness Questionnaire), the RAPA (RAPA1 and RAPA2) (Rapid Assessment of
Physical Activity), and the NHS (Nurses’ Health Study) sedentary lifestyle questionnaire.
All these questionnaires are described in the PREDIMED-PLUS website:
http://www.PREDIMEDPLUS.COM. Each participant randomly assigned to the
intervention group is also provided with a pedometer (Yamax SW200 Digi-Walker) to
self-monitor the number of steps walked per day. GENEActiv accelerometers are
provided as well to a subset of participants (50% of participants in the intensive

24
intervention group and 20% of those in the control group) in order to quantify physical
activity at baseline, 6 months, 1 year, and each year thereafter. In accordance with an
evaluation based on physical status, recommendations on aerobic physical activities and
strength training are progressively made and activities to improve balance and flexibility
encouraged on completion of physical activity.
Self-reported information during follow-up. At the follow-up visits, participants are asked
about clinical events that may have occurred between visits and information about
medication prescribed is updated.
Evaluation of adverse effects: At 6 months and yearly thereafter, participants complete
a specific questionnaire to report any adverse effects felt to be derived from the
intervention or weight loss.
Electrocardiogram: ECGs are performed at the primary care centers at the first screening
visit, 6 months, and at annual follow-up visits thereafter. The ECGs will be scanned,
stored and registered in the specific database designed for that purpose. The nursing
staff at each recruiting center will be responsible for receiving and scanning the ECGs,
digitizing their contents, and maintaining the registry and database.
Fasting blood collection: Fasting blood samples are collected at the baseline visits, 6
months, 12 months, 3 years, 5 years, 7 years, and at the final follow-up visit.
Conventional analyses [lipid profile (total cholesterol, HDL-cholesterol, LDL-cholesterol,
and triglycerides), fasting plasma glucose, blood cell count, serum sodium, potassium,
calcium, uric acid, urea, creatinine, albumin, C-reactive protein, erythrocyte
sedimentation rate, hemoglobin A1C, liver function tests (serum bilirubin, alkaline
phosphatase, alanine transaminase, aspartate aminotransferase and gamma-
glutamyltranspeptidase) and optional coagulation tests (prothrombin time, activated
partial thromboplastin time and fibrinogen)] are performed at the baseline and follow-up
visits according to the trial protocol. The nursing staff at the recruiting centers is
responsible for collecting, processing, delivering, storing and preserving the samples,
digitizing the information, and maintaining the registry and database for all samples and
analyses.
Morning spot urine collection: A sample of morning spot urine is collected in situ at the
baseline visit, 6 months, 12 months, 3 years, 5 years and 7 years, and at the final follow-
up visit. Conventional analyses (albumin and creatinine in urine) are performed at the
baseline and follow-up visits according to the trial protocol. The nursing staff is
responsible for collecting, processing, delivering, preserving, recording, and maintaining
the samples.

25
Nail sample collection: Nail samples are collected in situ at the baseline visit, 12 months,
3 years, 5 years and 7 years and at the final follow-up visit. Patients are asked to attend
the visits without having cut their toenails. Using toenail clippers, the patients’ nails will
be cut and placed in labelled zip-lock bags.
Neuropsychological and Quality of Life evaluation:
This evaluation includes three parts:
A) Cognitive Function
B) Quality of life
C) Psychopathology
At baseline and every two years thereafter (2-, 4-, 6- and 8-year follow-up visits),
participants complete a battery of 6 tests of Cognitive Function (see below, section A).
The first (Mini-Mental State Examination (MMSE) is a general screening cognitive test,
while the other 5 tests explore different cognitive domains and are aimed at assessin
changes in cognitive performance. The Cognitive Function tests will be alternated with
the Quality of life tests (see section B), so that in even years the 6 tests of Cognitive
Function will be collected and in odd years only the quality of life test will be administered
(see below, section B). The 2-year lapse between sequential cognitive tests will reduce
bias due to a "learning" effect.
The Quality of Life scales (Short -Form 36 or SF -36, see section B) are collected from
all participants at the beginning of the study and in odd years thereafter (after 1-, 3-, 5-,
and 7- year follow-up visits), while the psychopathology questionnaires (see below,
section C) are collected at the beginning of the study and annually.

All instruments included in the Cognitive battery (A), Quality of Life (B), and
psychopathology (C) have been standardized for the Spanish population in the age
range of the study. The complete battery of cognitive, quality of life, and psychopathology
examinations includes the following tests:

A) Six cognitive neuropsychological tests (lasting 16 minutes, to be completed in face-

to-face interviews):
1) MMSE (Folstein et al, 1975);
2) Semantic verbal fluency test: “animals in 1 minute” (Ramier and Hécaen, 1970, 1977;
Benton et al., 1994);
3) Phonemic verbal fluency test: “words in 1 minute starting with the letter ‘p’ (Benton et
al., 1994);
4) Verbal and visual working memory: reverse digits test (WAIS-III), Wechsler, 1997):

26
5) Trail Making Test (Reitan, 1973);
6) Clock test (Clock drawing test or CDT).

Normative data for these tests in the Spanish population have been published by Peña-
Casanova et al. (2009a, 2009b). These six tests are collected in the run-in period and
each even follow-up year thereafter (years 2, 4, 6 and 8). These six tests, which take
roughly 16 minutes to complete, are administered by PREDIMED-PLUS personnel at the
third screening visit and each even follow-up year thereafter (years 2, 4, 6 and 8).

B) One test of Quality of Life (5-10 minutes, to be completed at home):

The SF-36 (36-item) quality of life questionnaire (Alonso et al., 1995, 1998; Ware and
Gandek 1998) is administered during the run-in period and every odd year of follow-up
thereafter (years 1, 3, 5 and 7). In this way, these tests will be alternated with the
neuropsychological questionnaires.

C) Three Psychopathological questionnaires (lasting 20-25 minutes, to be completed at

home):
1) Beck Depression Inventory (BDI-II) (Beck, Steer and Brown, 1996; Sanz, Navarro
and Vázquez, 2003);
2) Multidimensional scale of weight locus control (Wallston, Wallston and DeVellis,
1978);
3) Screening for comorbid eating disorders with diagnostic criteria (DSM-IV-TR; APA,
2000).

The 10 questionnaires above (sections A, B and C) are collected in all participants in the
PREDIMED-PLUS study.
The four questionnaires of sections B) and C) are delivered to the participants at the
screening visit 1 to be completed at home or at another time outside the study visit.
Participants are required to deliver filled-in questionnaires to the recruiting centers within
a 15-day period. The same procedure is repeated at follow-up visits when required. The
nursing staff at each recruiting center is responsible for collecting, processing, sending,
and keeping all the information pertaining to the cognitive tests.
To ensure that graphical data from cognitive tests (drawings of the MMSE and clock and
Trail Making tests) are saved for future monitoring. The questionnaires from Group A, B
and C tests are collected in paper format, taking advantage of the optical scanning forms
for the MMSE, the clock test and the FS-36 designed for this purpose. Once completed,

27
the MMSE, the clock test and the SF-36 are mailed to the Navarra center (after saving a
security photocopy in the recruiting center) to be computerized by optical reading, a time-
saving and materially mistake-free procedure.

9. Outcome definition and ascertainment

Clinical events will be ascertained by a Clinical Event Ascertainment Committee led
by Dr. Fernando Arós of the Vitoria group. The committee members are M. Aldamiz, A.
Alonso, J. Berjón, L. Forga, J. Gállego, M. A. García Layana, A. Larrauri, J. Portu, J.
Timiraos, and M. Serrano-Martínez. Clinical event ascertainment will be based on
information collected from the participants’ medical records, which each year will be
reviewed on an ad hoc basis by the medical doctors participating in the PREDIMED-
PLUS trial. These doctors and the members of the Ascertainment Committee will be
blinded to the assignment of participants to the two intervention groups. The reports sent
to the Clinical Events Committee will contain no personal information about the
participants and will be identified only by a code.

9.1. Primary outcomes

1. Non-fatal acute coronary syndrome (acute myocardial infarction), non-fatal stroke or
cardiovascular mortality.
1.a. Acute myocardial infarction (MI) will be defined according to the third universal
definition of MI on behalf of the Joint ESC/ACCF/AHA/WHF Task Force (Thygesen et
al., 2012) as evidence of myocardial necrosis in a clinical setting consistent with acute
myocardial ischemia.

Any one of the following criteria meets the diagnosis for MI:
• Detection of a rise and/or fall of cardiac biomarker values [preferably cardiac
troponin (cTn)] with at least one value above the 99th percentile upper reference
limit (URL)
AND
• At least one of the following:
(i) Symptoms of ischemia.
(ii) New or presumed new significant ST-segment–T wave (ST–T)
changes or new left bundle branch block.
(iii) Development of pathological Q waves in the ECG.
(iv) Imaging evidence of new loss of viable myocardium or new
regional wall motion abnormality.

28
 (v) Identification of an intracoronary thrombus by angiography.
Prior MI
Any one of the following criteria meets the diagnosis for prior MI:
• Pathological Q waves with or without symptoms in the absence of non-ischemic
causes.
• Imaging evidence of a region of loss of viable myocardium that is thinned and fails
to contract, in the absence of a non-ischemic cause.
• Pathological findings of a prior MI

1.b. Stroke will be defined as an acute neurological deficit lasting more than 24 hours
caused by an abrupt impairment of brain function due to blockage of blood flow in a
particular artery supplying the brain (thrombosis or arterial embolism) or a cerebral
haemorrhage.

Ischemic Stroke is defined following the updated definition of stroke for the 21st
Century: A Statement for Healthcare Professionals from the American Heart
Association/American Stroke Association (Sacco RL, et al. 2013) as an episode of
neurological dysfunction caused by focal cerebral, spinal, or retinal infarction.
Central nervous system (CNS) infarction is brain, spinal cord, or retinal cell death
attributable to ischemia, based on:
1. Pathological, imaging, or other objective evidence of cerebral, spinal cord, or
retinal focal ischemic injury in a defined vascular distribution;
2. Clinical evidence of cerebral, spinal cord, or retinal focal ischemic injury
based on symptoms persisting ≥24 hours or until death, and exclusion of
other potential causes such as hypoglycaemia or seizures.
Silent CNS infarction will not be considered as a primary end-point if defined as
imaging or neuropathological evidence of CNS infarction without a history of acute
neurological dysfunction attributable to the lesion.

Haemorrhagic Stroke. Stroke caused by intracerebral hemorrhage is defined as

rapidly developing clinical signs of neurological dysfunction attributable to an
intracerebral hemorrhage, defined as a focal collection of blood within the brain
parenchyma or ventricular system that is not caused by trauma. Stroke caused by
subarachnoid hemorrhage is defined as a rapidly developing signs of neurological
dysfunction and/or headache because of bleeding into the subarachnoid space,
which is not caused by trauma.

29
 Silent cerebral hemorrhage will not be considered as primary end-point. It is defined
as a focal collection of chronic blood products within the brain parenchyma,
subarachnoid space, or ventricular system detected at neuroimaging or
neuropathological examination that is not caused by trauma and lacks a history of
acute neurological dysfunction attributable to the lesion.

1.c. Cardiovascular mortality: Includes sudden death and non-sudden cardiovascular

death (Buxton AE, et al. 2006).

Sudden (cardiac) death is due to cessation of cardiac activity with hemodynamic

collapse, typically due to sustained ventricular tachycardia/ventricular fibrillation. It
may be:
— Witnessed instantaneously in a previously stable patient. This may occur with or
without preceding signs or symptoms, or may occur immediately following sudden
dyspnea, light-headedness, or palpitations.
— Unwitnessed. Patient found dead who at the time of last witnessed contact was
in his/her usual state of health without medical complaints or obvious difficulty.
This applies to patients dying during sleep.

Non-sudden cardiac death: Includes deaths of patients from acute pulmonary

edema with severe, progressive heart failure, cardiogenic shock, or after a recent
cardiac surgical procedure.
Non-cardiac vascular death: Includes deaths due to thromboembolic events, stroke,
dissecting aneurysm and peripheral artery disease.

2. Weight change. The study nurse records weight at each follow-up visit. The
measurement are made according to the study manual of operations and with
participants dressed in light clothing and no shoes and accessories.

9.2. Secondary outcomes

1. Total mortality. This endpoint comprises all causes of death, including those from
CVD (see point 1c of primary end-point), as well as trauma, renal failure,
neoplasia, sepsis, suicide and death of undetermined cause. All deaths should be
confirmed by reviewing the National Death Index.

30
2. Changes in waist circumference. The study nurse will measure waist circumference
at each follow-up visit according to the manual of operations.

3. Non-ST-segment elevation acute coronary syndrome (unstable angina): The

diagnosis of unstable angina will be made following the definition of the ESC
Guidelines for the management of acute coronary syndromes in patients presenting
without persistent ST-segment elevation (Hamm et al, 2011); It requires the presence
of at least one of the following clinical characteristics:
a. Prolonged (>20 min) anginal pain at rest.
b. New onset (de novo) angina (Class II or III of the Classification of the Canadian
Cardiovascular Society).
c. Recent destabilization of previously stable angina with at least Canadian
Cardiovascular Society Class III angina characteristics (crescendo angina).

4. Coronary revascularization (percutaneous or surgical): The two main indications for

percutaneous or surgical revascularization are:
1) Patients with unstable angina or non-ST-segment elevation acute coronary
syndrome.
2) Patients considered likely to benefit from such surgery on the basis of the location
and severity of chest pain, the number of vessels affected, and the presence of
left ventricular dysfunction (Hamm et al, 2011).

5. Heart failure. Acute and chronic heart failure (HF) is a syndrome in which patients
have typical symptoms and signs resulting from an abnormality of cardiac structure
or function (McMurray JJ, et al. 2012; Yancy CW, et al. 2013). The cardinal
manifestations of HF are dyspnea and fatigue, which may limit exercise tolerance,
and fluid retention, which may lead to pulmonary and/or splanchnic congestion and/or
peripheral edema.
5.a. The diagnosis of HF with Reduced Ejection Fraction requires three conditions to
be satisfied: 1. Symptoms typical of HF; 2. Signs typical of HF and 3. Reduced
ejection fraction (< 40%)
5.b. The diagnosis of HF with Preserved Ejection Fraction requires four conditions to
be satisfied: 1. Symptoms typical of HF; 2. Signs typical of HF; 3. Normal or only
mildly reduced left ventricular ejection fraction and non-dilated left ventricle; and
4. Relevant structural heart disease (left ventricular hypertrophy/left atrium
enlargement) and/or diastolic dysfunction

31
 5.c. A Heart Failure event may include hospitalization or an urgent outpatient visit. In
this setting the event needs to meet ALL of the following criteria:
- The patient exhibits documented new or worsening symptoms of HF on
presentation, including at least ONE of the following: Dyspnea, decreased
exercise tolerance, fatigue or other symptoms of worsened end-organ perfusion
or volume overload.
- The patient has objective evidence of new or worsening HF, consisting of at least
TWO physical examination findings OR one physical examination finding and at
least ONE laboratory criterion), including: Physical examination findings
considered to be due to heart failure, including new or worsened peripheral
edema, increasing abdominal distention or ascites (in the absence of primary
hepatic disease), rales/crackles/crepitations at pulmonary auscultation,
increased jugular venous pressure and/or hepatojugular reflux, S3 gallop, and
clinically significant or rapid weight gain thought to be related to fluid retention
- Laboratory evidence of new or worsening HF, if obtained within 24 hours of
presentation, including: Increased B-type natriuretic peptide (BNP)/ N-terminal
pro-BNP (NT-proBNP) concentrations OR cardiological evidence of pulmonary
congestion OR echocardiographic data of congestion or decreased cardiac
output.
- The patient receives initiation or intensification of specific treatment for HF.

6. Peripheral artery disease. Ascertainment will be made according to the Inter-Society

Consensus for the Management of Peripheral Arterial Disease (TASC II) (Norgren
et al., 2007) and ESC Guidelines for the diagnosis of peripheral artery disease
(Tendera M, et al., 2011). For participants with intermittent claudication, aged 60-69
with one cardiovascular risk factor, or aged ≥70 years, a resting ankle-brachial
systolic pressure index ≤0.90 or an abnormal echo-Doppler examination, magnetic
resonance imaging, or arteriography will be considered as diagnostic (confirmed
case).
7. Venous thromboembolism (VTE): all VTE need to satisfy the standard diagnosis
criteria for venous thrombosis or (thromb-) Pulmonary Embolism (PE) in the general
population (see below 1-3). The diagnosis should be confirmed by objective imaging
techniques (including echography, phlebography, pulmonary computed tomography
angiography (angioCTA), NMR, etc.) and not only be based on the clinical suspicion.
Standard diagnosis criteria for VTE in clinical studies (Carrier M et al, 2012):
1. Deep venous thrombosis, defined as the loss of venous compressibility or the inability

32
 of filling the deep vein intraluminal segment at the lower/upper limbs, as detected by
echography with venous compression or phlebography, respectively.
• The presence of thrombus at the distal lower limb (distal from the popliteal vein)
qualifies for primary VTE only if it is asymptomatic.
• All proximal thrombus qualify for final primary end-point if detected by imaging
techniques (echography or radiology), regardless of whether it is or not asymptomatic.
2. Pulmonary Embolism (PE) is defined as:
Contrast pulmonary arteriography:
• Defects in intraluminal filling, as contrasted with two projections.
• Sudden stoppage of the contrast in one or several vessels with a diameter greater
than 2.5 mm
• Pulmonary scintigraphy based on ventilation/perfusion (V/Q):
o A V/Q-pulmonary scintigraphy with high probability of PE in patients with no low
clinical probability of PE.
• Pulmonary angiography using computed tomography:
o Defects in filling sub-segmental or more proximal vessels
3. Fatal PE is defined as:
• Death exclusively caused by PE and/or its confirmation at autopsy or using radiology
techniques
Important considerations:
a) Superficial venous thrombophlebitis should not be described as VTE.
b) It is highly recommended to describe VTE according to the anatomic position:
• Lower limbs
• Upper limbs
• Pulmonary embolism
• Others: vessels at the splanchnic level, cerebral veins, etc.
c) The description of the VTE is highly convenient (for instance, distal to popliteal vein
vs. proximal VTE; sub-segmental level vs. central PE)
d) VTE associated with a central catheter (for instance, deep venous thrombosis at the
upper limbs) should be reported separately.
e) Incidental VTE should be differenced from any other symptomatic events.

8. Atrial fibrillation (AF): AF is defined following the Guidelines of the American College
of Cardiology Foundation/American Heart Association Task Force on Practice
Guidelines together with the European Society of Cardiology, the European Heart
Rhythm Association, and the Heart Rhythm Society (Camm AJ, et al. 2010; Fuster V,

33
 et al. 2011), as a cardiac arrhythmia with the following characteristics:
(1) The surface ECG shows ‘absolutely’ irregular RR intervals, i.e., RR intervals that
do not follow a repetitive pattern.
(2) There are no distinct P waves on the surface ECG. Some apparently regular atrial
electrical activity may be seen in some EKG leads, most often in lead V1.
(3) The atrial cycle length (when visible), i.e., the interval between two atrial
activations, is usually variable and <200 ms (>300 bpm).

9. Type 2-diabetes. New-Onset Type 2 Diabetes cases are diagnosed following the
recommendations of the American Diabetes Association:
1. HbA1C ≥6.5%. This test should be performed in a laboratory using a method that
is National Glycohemoglobin Standardization Program (NGSP) certified and
standardized to the DCCT assay. OR
2. Fasting plasma glucose (FPG) ≥126 mg/dL (7.0 mmol/L). Fasting is defined as no
caloric intake for at least 8 hours OR
3. Two-hour plasma glucose (PG) ≥ 200 mg/dL (11.1 mmol/L) during an oral glucose
tolerance test (OGTT). This test should be performed as described by the WHO,
using a glucose load containing the equivalent of 75 g anhydrous glucose
dissolved in water OR
4. In a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a
random plasma glucose ≥200 mg/dL (11.1 mmol/L).
In the absence of unequivocal hyperglycemia, results should be confirmed by repeat
testing (American Diabetes Association, 2011).

10. Type-2 diabetes complications. Participants will be assessed yearly for

microvascular complications of diabetes:
1. Diabetic nephropathy: Kidney disease in diabetes is defined based on the
alteration of glomerular filtration rate (GFR) and /or the presence of persistent
albuminuria at levels of 30 mg/24 h or more (normal albumin excretion is currently
defined as < 30 mg/24 h). GFR is estimated through a quantitative formula, the
Modification of Diet in Renal Disease (MDRD) equation, that measures the
progression of kidney involvement. Persistent albuminuria is determined by the
urine albumin to creatinine ratio (normal <30 mg albumin/g creatinine) in a routine
morning urine sample. Because of variability in urinary albumin excretion, two of
three morning specimens collected within a 3- to 6-month period should be
abnormal before considering a patient to have developed increased urinary

34
 albumin excretion or a progression of albuminuria. The presence of one or two of
the above criteria indicates renal disease in these patients and the requirement
for appropriate follow-up for progression of renal disease (American Diabetes
Association, 2011).
The stages of chronic kidney disease by GFR will be reported as follows:
1. Kidney damage with normal or increased GFR ≥90 mL/min/1.73 m2 body
surface area
2. Kidney damage* with mildly decreased GFR 60–89 mL/min/1.73 m2 body
surface area
3. Moderately decreased GFR 30–59 mL/min/1.73 m2 body surface area
4 Severely decreased GFR 15–29 mL/min/1.73 m2 body surface area
5. Kidney failure <15 mL/min/1.73 m2 body surface area or dialysis
2. Diabetic retinopathy: Diagnosed by ophthalmologic examination and/or treatment
with laser photocoagulation (American Diabetes Association, 2011).
3. Diabetic polyneuropathy: Diagnosed by clinical symptoms, neurological
examination and results of electrophysiological studies of peripheral nerves
(American Diabetes Association, 2011).
*Kidney damage defined as abnormalities in urine, blood, or imaging tests.

11. Cancer. All cancers except non-melanoma skin cancer will be considered. Cancer
cases will be coded according to the International Classification of the World Health
Organization (International Agency for Research in Cancer, WHO, 2014).

12. Dementia/Alzheimer’s disease. Cases will be ascertained according to the

Recommendations from the National Institute on Aging and the Alzheimer´s
Association workgroup (McKhann et al., 2011) or if a diagnosis of dementia is
reported by a neurologist.

13. Other dementias: Cases will be ascertained according to McKhann et al. 2011
criteria (see below) or if a diagnosis of dementia is reported by a neurologist.
Dementia is diagnosed when there are cognitive or behavioral (neuropsychiatric)
symptoms that: 1. Interfere with the ability to function at work or at usual activities;
and 2. Represent a decline from previous levels of cognitive functioning; and 3. Are
not explained by delirium or major psychiatric disorder; 4. Cognitive impairment is
detected and diagnosed through a combination of (1) history-taking from the patient

35
 and a knowledgeable informant and (2) an objective cognitive assessment; 5. The
cognitive or behavioral impairment involves a minimum of two domains).

14. Parkinson’s disease. Cases will be ascertained according to the diagnostic criteria
described by Hughes et al (Hughes AJ, et al., 1992) or if reported by a neurologist.

15. Unipolar depression. The diagnosis should be made according to the DSM-V criteria
(American Psychiatric Association, 2013). In this, definition major depression,
persistent depression, and other depressions included in Depressive Disorders
(DSM V) are accepted. Diagnosis of depression made by primary care physicians
or psychiatrist in participants treated with antidepressant drugs for more than 6
moths will be accepted. If this is the case, ICD 10 (International Statistical
Classification of Diseases and Related Health Problems, 10th version) diagnosis of
depressive episodes are also accepted. For physicians and psychiatrists not using
ICD 10 or DSM V, a positive response to the two questions included in the NICE
clinical guidelines is recommended (https://www.nice.org.uk).

16. Osteoporotic fractures. Low-energy fracture is defined as the fracture produced by

a same-level fall. Fractures will be identified from X-rays reports obtained from at
least two radiological reports. High trauma fractures, potentially pathological
fractures (e.g., cancer or Paget’s disease), or fractures of the head, fingers and toes
will not be considered (Bliuc D, et al. 2009).

17. Gallstone disease or cholecystectomy: Gallstone disease will be diagnosis according

to the findings obtained by imaging techniques including abdominal
ultrasonography, computed tomography or magnetic resonance imaging. Diagnosis
of cholecystectomy will require the corresponding surgical report.

18. Symptomatic gout: Defined following the criteria of the American College of
Rheumatology. Typically the disease first presents as arthritis that is acute and
episodic, but can be recurrent. Gout can also present as chronic arthritis of one or
more joints. This clinical picture is built on a foundation of an excess body burden of
uric acid, manifested in part by hyperuricemia, which is defined as serum uric acid
levels greater than 7.0 mg/dL (Khanna D, et al. 2012).

36
19. Transient Ischemic Attack: The diagnosis should be made according to the Scientific
Statement of the American Heart Association/American Stroke Association Stroke
Council (Easton JD et al. 2009): a transient episode of neurological dysfunction
caused by focal brain, spinal cord, or retinal ischemia, without acute infarction
demonstrated by neuroimaging, preferably magnetic resonance imaging
techniques.

20. Cataracts surgery: Defined by a medical report of cataracts surgery.

21. Surgery for obesity: Defined by a medical report of bariatric surgery.

9.3. Intermediate markers

Changes in nutrient intake and dietary patterns will be determined by changes in the
17-item score of adherence to the energy-restricted Mediterranean diet (intensive
intervention group) or the 14-item score (control group) and by changes in food and
nutrient intake determined by the 143-item food frequency questionnaire administered
during follow-up.
Changes in systolic and diastolic blood pressure, serum lipid concentrations, fasting
glucose levels, renal function, uric acid, hemoglobin A1C, C-reactive protein, and liver
function will be evaluated yearly for the duration of the intervention.
Also evaluated yearly will be the percentage of participants in each group requiring
anti-hypertensive, anti-diabetic or lipid-lowering medication, results of ECGs, cognitive
function, quality of life, and psychological and neuropsychological questionnaire scores.

10. Randomization procedure (random assignment)

Between one and four weeks after the third screening visit, each recruiting center
will randomly assign eligible candidates to one of two groups, intensive intervention
group or usual care (control) group, using a centrally-controlled, computer-generated
random-number system (available at: www.predimedplus.com). The coordinating center
will be responsible for the randomization procedure by which participants will be
randomly assigned with stratification by center, sex, and age group (<65, 65-70, >70
years). Married or unmarried couples are randomized together. The recruiting centers
enter the participants’ identification criteria into the internet-based system. The system
then automatically assigns the participants or partners of participants to their groups.

37
Once this occurs, the group assigned cannot be changed. In the specific cases of
couples in which the spouse was recruited at different times, the last spouse entering
the study will be assigned (not randomised) to the same study arm than his/her partner
in order to ensure high adherence to the intervention and avoid contamination and
potential conflicts between partners of the same household.

11. Intervention protocol

All participants will continue to receive usual healthcare from their family doctors and
primary care physicians throughout the duration of the trial. At no time will PREDIMED-
PLUS personnel deliver medical care.

11.1. Phases of the study for participants assigned to the intensive intervention program
with energy-restricted Mediterranean diet (intervention group)

First six months

In addition to the initial visit, participants assigned to the intensive intervention
program will take part in six individual sessions (I) and 6 group sessions (G) in the first
six months (see below):

Month 1 Month 2 Month 3 Month 4 Month 5 Month 6

G I G I G I G I G I G I

Participants will also receive a third monthly contact by way of a telephone call
from a dietitian aimed at reinforcing the trial’s objectives and answering any queries.
During these first six months, participants in the intervention group are
encouraged to aim for a reduction in their initial weight of 10% and a reduction in their
initial waist circumference of between 5 and 10%. The aim of the trial during these first
six months is that the average weight loss of the participants in the intervention group is
above 8% and the average waist circumference reduction is above 5%. Success in
achieving an initial weight loss is known to be a predictor of long-term weight loss. For
this reason, all participants will be given a chart on which to record and correctly monitor
their own weight and waist circumference.
During this period they will be encouraged to substitute one meal for low-calorie
foods and so will be offered a wide range of pleasant alternatives in keeping with the
culinary traditions of the Mediterranean diet (see below).

38
 Finally, if by the final visit the participant has still been unable to reach the
objectives established for this phase of the trial (month 6), he or she will take part in a
motivational interview session with the dietitian in order to determine why he/she has
not reached his or her weight-loss goal (see below), try to readdress the situation, and
provide appropriate rescue measures.

Months 7-12
Participants will attend one individual session (I) and one group session (G) every
month in months 7 to 12 of the trial.

YEAR 1 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12

Months 7-12 G I G I G I G I G I G I

They will also receive a third contact every month by way of a telephone call
from a dietitian aimed at reinforcing the trial’s objectives and answering any queries.
The first-year follow-up visit (see below) will coincide with the last individual visit
for this phase.

Years 2-6
After the first year and in each of the remaining years of the trial (years 2-6), the
participants will attend one quarterly individual session (I) and one monthly group session
(G) and will receive two quarterly telephone calls (T), in accordance with the table below:

Mont Month Month Mont Month Month Mont Month Month Mont Month Month Mont
h 13 14 h 15 16 17 h 18 19 20 h 21 22 23 h 24

13- G T G T G I G T G T G I G T G T G I G T G T G I
72

Months 25-36 will follow the same procedure as months 13-24, and this procedure
will be repeated for the successive years.
The annual follow-up visits (see below) will coincide with the last individual session
of each year (month 24 above). Throughout the trial, any missed visits will be
reprogrammed.

39
11.2. Program of individual and group sessions for participants assigned to the energy-
restricted Mediterranean diet
A) Individual visits
All individual visits comprise:
i) Distribution of a 17-item questionnaire of adherence to an energy-restricted
Mediterranean diet.
ii) Weight and waist circumference measurement by a dietitian.
iii) An individual motivational interview with the dietitian in accordance with the changes
in weight observed and the participant’s scores on the 17-item adherence to
Mediterranean diet questionnaire (see below).
iv) Encouragement to self-monitor weight and waist circumference. Participants are
provided charts for self-registering and self-monitoring weight and waist
circumference in accordance with the Body Weight Simulator of the National Institute
of Diabetes and Digestive and Kidney Diseases (Hall et al., 2011; National Institute
of Diabetes and Digestive and Kidney Diseases, 2012). This simulator is also
provided to participants at the first individual session with use instructions.
v) Personalized recommendations for increasing physical activity.
B) Group sessions
At these group sessions participants are provided shopping lists, menus, recipes,
descriptions of typical components of the Mediterranean diet and advice on lifestyle
changes. PREDIMED-PLUS dietitians lead these sessions, which are attended by no
more than 20 participants. The sessions comprise:

i) An introductory talk to review the 17-item questionnaire on adherence to the energy-

restricted Mediterranean diet (see below).
ii) A 15-minute presentation of the main aspects of the Mediterranean diet with
audiovisual material prepared by the coordinating center.
iii) Answers to any queries on any aspect of the intervention.
iv) Delivery of the following documents:
• Description of 4-5 low-calorie foods typical of a Mediterranean diet and adapted to
the season.
• Weekly food shopping list adapted to the season.
• Weekly meal plan (with detailed menus) adapted to the shopping list.
• Recipes for the suggested menus.
v) Delivery of gratis virgin olive oil (one liter per month) and nuts (500 g per month) to
each participant.

40
vi) At the end of the session participants are reminded of the date of the next session.
11.3. Program of individual and group sessions for participants assigned to the control
group.
Control group participants receive usual medical care from medical staff at their health
institutions. The importance of their attending usual medical visits will be stressed to
them. Participants receive all the written information related to the Mediterranean diet
used in the PREDIMIED trial as well as leaflets with general lifestyle recommendations
for managing the metabolic syndrome. At the beginning of the study, a group session
and an individual session is held at which dietitians deliver documents similar to those
used in the PREDIMED trial (shopping lists, recipes, menus, and descriptions of
Mediterranean diet components). The dietitians do not provide participants in the control
group with instructions on how to lose weight, as this is the responsibility of their family
doctors or specialists (usual care). They are also offered a group session every 6
months. At the initial visit and at each 6-month group session, participants are provided
free virgin olive oil (6 liters every 6 months) and nuts (3 kg every 6 months). In order to
encourage compliance with the trial, supply of olive oil and nuts to the participants is
contingent on their attending these sessions. The 6-month group sessions include tips
on how to follow the Mediterranean diet to prevent CVD but advice on calorie restriction,
weight loss or increased physical activity is not given and no such objectives are
entertained.

11.4. Dietary and lifestyle intervention

The Intervention Committee led by Jordi Salas-Salvadó coordinates the dietary and
lifestyle intervention. This committee is made up of four coordinators (Jordi Salas-
Salvadó, Montse Fitó, Ramón Estruch and Miguel Ángel Martínez-Gonzalez), three of
whom will be responsible for the three intervention sub-committees: Dietary Intervention
(chair: Jordi Salas-Salvadó; members: Nancy Babio, Emilio Ros and Ana Sánchez-
Tainta); Physical Activity (chair: Montse Fitó; members: Helmut Schröder, Ascensión
Marcos, Miguel A. Martínez-González, Dolores Corella, and Julia Warnberg); and
Behavior Treatment (chair: Ramon Estruch; members: Fernando Fernández-Aranda,
Cristina Botella and Jordi Salas-Salvadó). This Committee is responsible for designing
the lifestyle intervention program for the intensive intervention group and ensuring that it
is implemented correctly. Miguel Ruiz-Canela, Miguel A. Martínez-González and Jordi
Salas-Salvadó are responsible for ethical considerations.

41
Dietary recommendations
Many aspects of a diet’s quality can affect body weight and the risk of obesity-related
illnesses to a greater extent than relative macronutrient content (Mozaffarian et al., 2011;
Ludwig, 2012). In recommendations given to participants, two food groups (A and B) will
be clearly differentiated:
A) Traditional dietary patterns based on whole foods or minimally processed foods, such
as the Mediterranean diet, which incorporates many cardioprotective foods and few
harmful ones. The consumption of virgin olive oil, nuts (especially walnuts), fruits and
vegetables, salads, whole grains, fiber-rich foods and low-fat yogurts have been
consistently associated with weight loss or lower weight gain (Martinez-Gonzalez,
Bes-Rastrollo, 2011; Mozaffarian et al., 2011).
B) On the other hand, sugar-sweetened beverages, fast foods, refined grain products
(especially white bread, which is widely consumed in Spain), white rice, pasta (except
for whole-grain pasta), French fries, potatoes, trans fats (mainly present in
commercial bakery products in Spain), sweets, cakes, pies, sugar, precooked meals,
sausages or cold cuts of processed meats, and patés have been consistently
associated with weight gain (Schulze et al., 2006; Mozaffarian et al., 2011).

The main focus of the intensive intervention program (intervention group) lies
therefore in the diet’s overall quality, with the aim of avoiding foods from the B group and
replacing them with foods from the A group.
In addition, by taking into account energy requirements estimates according to the
Institute of Medicine equation as well as the participants’ basal metabolic rate and level
of physical activity, a reduction in energy intake of roughly 600 kcal (about 30% of
estimated energy requirements) is envisaged. The energy-restricted Mediterranean diet
involves reduced consumption of meat and cold cuts, sugars, white bread, processed
fruit juices and sugary beverages, and other foods from the B group, as follows:

ENERGY-RESTRICTED MEDITERRANEAN DIET

NUTRIENT RECOMMENDED INTAKE
Calories1 Reduction of ≈600 kcal/day (about 30%) from usual
intake
Total fat2 35-40 % of total calories
Saturated Fatty Acids 8-10 % of total calories
Monounsaturated Fatty Acids > 20 % of total calories
Polyunsaturated Fatty Acids > 10 % of total calories
Cholesterol3 < 300 mg/day
Proteins4 Approximately 20 % of total calories
Carbohydrates5 40-45 % or more of total calories (of low glycemic

42
 index)
Sodium chloride No more than 100 mmol/day (roughly 2.4 g of sodium
or roughly 6 g of sodium chloride)
Dietary fiber 30-35 g/day

1. A reduction in calories of 500 to 1,000 kcal/ day will help to achieve a weight loss of
0.5 to 1 kg/week.
Alcohol provides unnecessary calories and displaces the intake of more nutrient-dense
foods. Not only does the consumption of alcohol increase the number of calories in one’s
diet but in epidemiological and experimental studies it has also been associated with
obesity. For this reason, although the 17-item adherence to the Mediterranean diet
questionnaire contains one item for the consumption of wine, the impact of calories from
alcohol on the overall calorie intake should be carefully evaluated and monitored and the
consumption of alcoholic beverages other than wine should be avoided.
2. The consumption of wine permitted is one or two glasses per day for women and two
or three glasses per day for men. The consumption of other sources of alcohol other than
wine is discouraged. Red wine is preferred over other types and it is recommended that
the wine be consumed at mealtimes.
3. Fat restriction involves fat from animal foods. Olive oil and nuts must be the preferred
sources of fat.
4. Proteins should be derived first from plant and second from lean animal sources (like
fish or poultry).
5. Carbohydrates should be derived from solid, minimally processed and fiber-rich foods
with a low glycemic index, such as vegetables, fruits and whole grains, all of which are
good sources of vitamins, minerals, and fiber. A diet that is rich in soluble fiber such as
oat bran, legumes, and most fruits and vegetables may be effective in reducing blood
cholesterol levels and insulin resistance. A diet that is high in all types of fiber may also
help to control weight by promoting satiety and maintaining lower levels of total energy
intake.
6. During weight loss, attention should be given to maintaining an adequate intake of
vitamins and minerals. Maintaining the recommended calcium intake of 1,000 to 1,500
mg/day is especially important for postmenopausal women who may be at risk of
osteoporosis.

43
 Participants in the intensive intervention group receive counseling to help them
progressively increase their compliance with the following 17 objectives (the 17-item
questionnaire on adherence to the energy-restricted Mediterranean diet). One
point will be awarded for each objective met:

1. Use only extra-virgin olive oil for cooking, salad dressings, and spreads.
2. Consume ≥3 portions of fruit per day.
3. Consume ≥ 2 portions of vegetables/garden produce per day (at least 1 portion raw
or in a salad).
4. Reduce consumption of white bread to d1 serving/day (1 serving = 75 g).
5. Consume whole grain cereals and pasta ≥5 times per week.
6. Consume d1 serving (1 serving = 100-150 g) of red meat, hamburgers, or meat
products (ham, sausage, etc.) per week.
7. Consume less than 1 serving of butter or cream per week (1 serving = 12 g).
8. Consume less than one sugary beverage or sugar-sweetened fruit juice per week.
9. Consume ≥3 servings of legumes per week (1 serving = 150 g).
10. Consume ≥3 servings of fish or shellfish per week (1 serving = 100-150 g fish, or 4-
5 units or 200 g shellfish).
11. Consume < 3 sweets or pastries, such as cakes, cookies, sponge cake, or custard,
per week.
12. Consume ≥ 3 servings of nuts (including peanuts) per week (1 serving = 30 g).
13. Consume chicken, turkey or rabbit meat instead of beef, pork, hamburgers or
sausages.
14. Use sofrito (sauce made with tomato and onion, leek or garlic, simmered in olive oil)
≥ 2 times per week.
15. Do not add sugar to beverages (coffee, tea); instead, replace sugar with non-caloric
artificial sweeteners.
16. Reduce consumption of pasta or rice <3 servings per week (unless the pasta or rice
are whole grain products).
17. Consume 2-3 glasses of wine (200 mL) per day (men) or 1-2 glasses of wine per day
(women). This item will be only promoted among participants who were consumers of
alcohol at baseline. Abstainers will never be invited to start consuming wine or any other
alcoholic beverage.

The intervention tool for the control group, on the other hand, is the PREDIMED 14-
item adherence questionnaire to the non-energy-restricted Mediterranean diet

44
(Schroeder et al., 2011). However, the 17-item questionnaire is also collected in control
group participants for comparison purposes.

Physical exercise recommendations

Participants are encouraged to gradually increase their level of physical activity to at
least 45 minutes per day (6 days per week) after 6 months of intervention and their
progress is monitored. The physical activity program includes aerobic activities, such as
gentle walking or any equivalent activity of moderate intensity and resistance training
(Fernández et al., 2012). The dietitians adapt their recommendations to personal
preferences and encourage participants to switch between activities with the same
metabolic equivalence of tasks.

Psycho-behavioral therapy
Participants are instructed on strategies and provided tools for solving problems
associated with consuming high calorie foods and performing sedentary activities. They
are encouraged to learn how to recognize lack of control on food intake under stressful
or anxious situations and how to exercise self-control.

Recommendations on the use of tobacco

The PREDIMED-PLUS dietitians will make no recommendations on the use of tobacco.
This is the responsibility of the medical professionals in the primary care centers in
accordance with usual medical practice.

Individual motivational interviews

Personal interviews with the dietitian at each individual visit are adapted to the
participant’s clinical conditions, preferences and beliefs. Dietary changes are introduced
in order to achieve the recommended diet for each participant and suitable lifestyle
changes are incorporated. Objectives are accorded via a negotiated agreement between
the two parties (dietitian and participant) depending on what participants consider to be
an attainable goal. The main objective is to change not only the participant’s consumption
of certain foods but also his or her overall dietary pattern. Attention can vary between
changing portion sizes, changing the frequency of dietary components, and changing
cooking methods.
Achievements made in the previous months, however minor, are always considered
an essential support mechanism for improving self-esteem and self-reward. Special care

45
is taken to ensure that participants do not receive contradictory dietary advice from health
professionals external to the PREDIMED-PLUS trial.
As described, each participant receives oral and written information on the food
components and culinary customs of the energy–restricted Mediterranean diet, as
well as charts for self-registering and self-monitoring changes in weight and waist
circumference at each visit.
Participants who during the active weight-loss phase have observed a lower weight
loss than expected or who have not maintained the weight loss they had achieved
receive special reinforcement and a series of rescue measures to help them achieve
weight loss and weight-loss maintenance. In such cases, agreements are negotiated
between the dietitians and the participants.

Role of the dietitians

The PREDIMED-PLUS dietitians are directly responsible for the dietary intervention.
They have been specifically trained and certified to deliver the PREDIMED-PLUS
intervention protocol. All intervention procedures are conducted in accordance with the
PREDIMED-PLUS operation’s manual. Throughout the study, annual meetings will be
held at which the dietitians will discuss any problems they may have identified and find
possible solutions. The dietitians and trial coordinators will discuss any problems arising
during the trial, thus ensuring a process of continuous feedback.

12. Training and calibration procedures

A general trial operations manual and staff training documents are set forth to ensure
standardized procedures across the various recruiting centers. Before implementation of
the protocol, study personnel attended a 3-day training course at the coordinating center.
This included theoretical and practical group discussions with experts on lifestyle
interventions in order to convey the goals of the study, develop all the specific aspects
involved in implementing the intervention, and impart training on the informed-consent
process, anthropometric and blood pressure measurements, data collection by optical
scanning or online systems, and biological sample collection and processing. The
abilities of all contracted personnel were evaluated at personal interviews during this
training course. The research team stressed the importance of creating a trusting and
empathic relationship with the participants and paying attention to their individual needs
in order to maximize their motivation and retention into the trial. Study personnel keep a
copy of the operations manual detailing all the training points. In addition, all the
personnel responsible for the intervention will attend annual meetings and be in constant

46
contact with the principal investigators in order to ensure standardized implementation
of the trial protocol. In accordance with the protocol, all scales and other measurement
instruments will be periodically calibrated.

13. Retention and compliance with strategies and supervision procedures

The recruitment of participants and the compliance rates for the intervention
strategies are crucial to the success of a trial of this nature. For this reason, a run-in
(pre-evaluation) period prior to randomization was planned: only participants who
adhere to all the requirements of the protocol during the run-in period are accepted into
the trial. The lag time between the end of the run-in period and the start of the intervention
ranges from one week to one month.
The researchers involved in this trial have already gained invaluable experience in
managing long-term trials through the PREDIMED trial, wherein they developed
strategies for ensuring participants’ compliance with the protocol and encouraging their
long-term retention. We understand that this is a particularly sensitive aspect for
participants in the control group. Therefore, at each group session of both the intensive
intervention group and the usual care group, virgin olive oil and mixed nuts is provided
at no cost to all participants. Our experience in the PREDIMED trial showed that such
gifts, especially the virgin olive oil, greatly helped to encourage participants’ retention.
Other retention strategies include providing feedback on findings during follow-up to the
participants’ usual health-care providers as well as supplying other non-coercive material
incentives for both groups. Additionally, in the intensive intervention group, where
significant weight-loss is anticipated, contact with participants is ongoing and flexible
interventions and rescue measures tailored to the participants’ needs are implemented.
Self-control, self-reward and self-monitoring techniques will also reinforce participants’
compliance with the intervention. Finally, the intervention is adapted to the needs of the
participants, which should encourage compliance.

14. Biological samples and laboratory procedures

The nursing staff contracted at each recruiting center is responsible for collecting,
processing and storing the biological samples in freezers at a temperature of -80ºC.
Blood samples are collected at the recruiting centers in the same way as they are
collected at the participants’ usual healthcare centers. In addition, 55.5 ml of blood are
extracted and collected in the following tubes: two 10 ml K2E EDTA tubes; one 4.5 ml
citrate tube; and two 10 ml and 6 ml gel serum separator tubes. The serum, citrate
plasma and EDTA plasma samples are distributed in aliquots of 200 μl and 500 μl and

47
stored at -80°C for future analyses at the recruiting centers. For the intensive intervention
group, the biochemical measurements will be performed in a blind fashion and in the
same batch for consecutive samples of each participant. Each recruiting center has an
ultra-low-temperature freezer with enough capacity to store biological samples until final
delivery. All biological samples are processed at each recruiting center no later than one
hour after extraction. During transportation from the primary care centers to the
laboratories, the biological samples are stored at 4ºC in a portable cooler. Urine tests
are conducted at the recruiting centers in the same way they are conducted at the
participants’ usual healthcare centers in accordance with the specific PREDIMED-PLUS
protocol for collecting biological samples.

15. Quality control

The general database for the PREDIMED-PLUS trial will be managed and
maintained by the research group of the IMIM Institute (CIBERobn). The food-frequency
questionnaires and the food records, as well as quality of life test, clok and Minimental
tests are processed and managed at the University of Navarra. Data collected from
accelerometers to measure physical activity are processed at the Malaga recruiting
center in collaboration with the CSIC/UAM. These data are sent every three months to
the IMIM, where they are incorporated into the General Database. Event detection data,
collected from information gathered during the intervention and at the follow-up visits,
will be introduced into specific forms at the recruiting centers, preferably using online
systems, and sent at least once a month to the data manager at the IMIM, who will send
monthly reports of missing or inappropriate entries back to the recruiting center
coordinators to solve any raised queries. The IMIM also sends monthly reports to the
different recruitment centers with the sumsample of each node. The steering committee
has been set up to ensure the quality of the project and correct any flaws or divergences.
This committee is made up of Jordi Salas-Salvadó (PREDIMED-Plus coordinator),
Miguel Angel Martínez-González, Ramón Estruch, Montserrat Fitó, Emilio Ros, and
Dolores Corella. At every annual PREDIMED-PLUS meeting, the IMIM will conduct a
current data management information session. An annual summary will be sent to the
recruiting center coordinator for distribution to all groups.
To reduce data entry expenses and speed up processing, the questionnaires and
data forms are processed by optical scanning or by online data transfer forms. The data
forms are entered in duplicate and missing data checks are performed. All forms sent to
another recruiting center must be photocopied and stored at that center. After data entry,
cross-form edit checks are performed and any data inconsistencies are identified. To

48
detect any still-unsolved problems, audits will be run periodically at each recruiting
center. Reports will be drafted to summarize any problems in the database and provide
an additional step to ensure the quality and accuracy of the data. To minimize the
possibility of error, a detailed operations manual has been prepared.
Annual staff training meetings will be conducted. The data manager and an audit
committee will evaluate the performance of each recruiting center. Appropriate new
procedures and corrective measures will be implemented whenever deficiencies are
noted. Until the end of the trial, all field centers will be masked to the trial outcome data
except for the two trial statisticians, one in Navarra (M.A. Martínez-González) and one
at the IMIM in Barcelona (Joan Vila), who will always perform the statistical analyses in
duplicate with two statistical analysis units. Because of the nature of the trial, however,
the dietitians at each field center know which intervention has been assigned to each
participant. The medical doctors who will prepare the annual report on the ad hoc review
of the participants’ medical records will be blinded to group assignment, as will the
Clinical Event Ascertainment Committee. The members of the Steering Committee,
who will attend the meetings of the Data and Safety Monitoring Board, will also remain
blinded to the results of intermediate analyses throughout the trial. The Steering
Committee will be informed of the total number of events observed but not of the groups
in which they occurred.

49
STATISTICAL ANALYSIS PLAN
All analyses will be performed on an intention-to-treat basis. Miguel A. Martínez-
González will be the senior statistician responsible for the statistical analysis plan. All
major data analyses will be conducted under his supervision. Statistical analyses for the
main aims of the study will be also conducted in duplicate by the center at IMIM,
Barcelona (responsible statistician at IMIM: Joan Vila).

1. Analysis of the effect of the intervention

Since the data take into account time to the event, Cox’s regression models will be
used to determine the effect of the intervention on the incidence of cardiovascular events.
For changes in weight and waist circumference, mixed models of analysis of variance
and generalized estimating equations (GEE) will be used. These models will include the
following adjustment covariates:
1) All factors that, according to the scientific literature, are related to the event; and
2) All factors that reach statistical significance in univariate analyses.

In these models we will evaluate: 1) the proportional hazards assumption; 2) the linearity
of the continuous variables, using smoothing spline methods; 3) the effect of extreme
observations on the estimation of parameters, by calculating delta-beta values. The use
of further approaches (i.e. normalizing transformations, stratified analyses, etc.) will
depend on the results obtained above. Given that participants will be clustered by
recruiting centers, some degree of correlation structure may be expected. Center will
therefore be included as a stratification variable, including frailty estimates, in the Cox
regression models. The goodness-of-fit of the models will be examined using the
modified Hosmer-Lemeshow test for survival studies. Robust estimators of variance that
account for the clustering effect of members of the same household (the second member
is not randomized for feasibility reasons) will be used to take into account the intra-cluster
correlation. Sensitivity analyses will be conducted after excluding the second (non-
randomized) members of the same household. In addition to the stratification by center,
all Cox models will be also stratified by sex and educational level.

2. Interim analyses and stopping rules

Data from the PREDIMED-PLUS trial will be analyzed after 3 years of median follow-up,
after 5 years of median follow-up, and at the end of the trial. For methodological reasons
but especially for ethical ones, suitable follow-up for a trial must include at least one
interim analysis (Schulz, Grimes, 2005). However, to preserve an overall alpha error of

50
0.05, interim analyses have to be penalized. We will use the O’Brien and Fleming
boundaries (O'Brien and Fleming, 1979). With this method, the boundaries are stricter at
the earlier stages of the study than at the later ones. Applying this rule leads to the
following p values for stopping the trial:
First interim analysis (median follow-up: 3 years); threshold p value: 0.0005.
Second interim analysis (median follow-up: 5 years); p value: 0.014.
Final analysis (median follow-up: 8 years); p value: 0.045

These p values should not be considered compulsory for stopping the trial but guidelines
for guaranteeing the security of the data. In making their decision, the Data and Safety
Monitoring Board must take into account, for example, the size of the effect, the follow-
up time at each recruiting center, the heterogeneity between the effects at the recruiting
centers, as well as evidence from other current trials and observational studies. All the
above must be taken into consideration when deciding either to continue or to interrupt
the trial after each interim analysis. Reasons for interrupting the trial include: 1)
convincing evidence of the beneficial effect of the intervention (the trial will be stopped
only if the effect of the intervention is great); 2) convincing evidence of a harmful effect
from the intervention; 3) results suggesting it is highly unlikely that the proposed
hypothesis will be accepted due to, for example, a very small effect of the intervention
that dramatically affects the trial’s statistical power.

3. Estimations of sample size

We will determine the effect of the intensive weight-loss lifestyle intervention with an
energy-restricted Mediterranean diet on the two primary outcomes below, assuming a
two-tailed alpha error of 0.05.

1. Effect of the intervention on the incident CVD (non-fatal myocardial infarction, non-
fatal stroke, and cardiovascular death). The cumulative projected incidence after
including as primary events all non-fatal acute myocardial infarctions and all
microinfarctions with positive high-sensitivity troponin tests after 6 years will be at least
10% in the control group, if we take into account the results of the PREDIMED trial after
4.8 years (which did not include high-sensitivity troponin tests). The hazard ratio (HR) for
the combined primary endpoint is anticipated to be 0.70 (Estruch et al., 2013) and will
probably be even lower (greater protective effect) if we consider that in the PREDIMED
trial no energy restriction was implemented, physical activity was not encouraged, and
weight loss was not a target of the intervention. Under these assumptions, therefore,

51
even if the dropout rates were to reach 20%, the required sample size would be 2,400
per group (see Figure 3). To be conservative, however, we aim to recruit 6,000
participants and assign 3,000 participants to each group. The participants will be
recruited at 20 recruiting centers, each of which will have the goal of recruiting, educating
and following approximately 300 participants, 150 of whom will be in the control group
and 150 in the intensive intervention group.

FIGURE 3. Estimation of the sample size required per intervention group in the
PREDIMED-PLUS trial

2. Effect of the intervention on weight change. Based on previous studies, we can

expect a minimum weight change for participants in the control group and a weight loss
of 3-4.5 kg for those in the intensive lifestyle intervention group, with a standard deviation
of 8 kg (Shai et al., 2008; Sacks et al., 2009; Wing, 2010). If we assume our intervention
will have only a small effect on weight change and then calculate sample size according
to a weight change of 1 kg in the usual care group, a weight change of 3 kg in the
intensive lifestyle intervention group, and a standard deviation of 8 kg, in order to achieve
a statistical power of 0.80 we would need a sample size of only 337 in each group. Since
the number of participants to be recruited is much higher than this figure, the statistical
power needed to reach this objective is largely guaranteed.

52
STRENGTHS AND LIMITATIONS
1. Strengths
A. This trial provides a multidisciplinary approach to tackling the serious problem
presented by the overweight and obesity epidemic. Our target group comprises obese
or overweight adults, who represent an increasing proportion of the general population.
For these subjects, an intervention based on a profound lifestyle change incorporating
improvements in the dietary pattern, weight loss, behavioral therapy, and increased
physical activity can be a novel and useful model for reducing the burden of obesity and
associated diseases, thus contributing to the sustainability of the healthcare system. The
trial clearly addresses priority objectives of the public healthcare system since it tackles
both the principal epidemic of our times (overweight and obesity) and the principal cause
of death around the world (CVD).
B. This innovative proposal presents a novel paradigm for nutritional recommendations
aimed at achieving weight loss, i.e., a traditional dietary pattern characterized by a
moderate-to-high fat content. We believe this new approach will help improve
compliance with the intervention and overcome the main challenge of any dietary
interventions aimed at fighting overweight and obesity: long-term weight loss
maintenance (Shai et al., 2008; Beunza et al., 2010; Romaguera et al., 2010).
C. The intervention is well structured and the trial is suitably designed for determining
the effect of the intervention on the main clinical outcomes. Moreover, since the trial is
conducted in the context of primary healthcare and incorporates epidemiological, clinical
and basic aspects, it has a high capacity for both transferability and reproducibility.
D. The research team includes investigators with invaluable experience in lifestyle
intervention trials (e.g., PREDIMED). As these investigators come from a wide range of
fields, their work will be complementary and the trial’s chances of success will be
enhanced. All these reasons, together with the success and achievements of the
PREDIMED trial, attest to the viability of this trial proposal.
E. From a strategic perspective, this is a timely proposal since it provides continuity for
the collaborative project in which most CIBEROBN centers have participated, i.e.,
PREDIMED.

2. Limitations
A. Our study will enroll participants aged between 55 and 75 years old. This may preclude
generalization to younger age groups.
B. In a large-scale clinical trial, one limitation to consider is participants’ dropout rates.
However, we hope to ensure compliance in both groups by: a) providing free foods (olive

53
oil and mixed nuts); b) establishing personal relationships with each participant via
individual and group sessions; c) administering, at the start of the study, the Prochaska
and DiClemente Stages of Change Model, by which a low predicted probability of
changing dietary habits will be a criterion for exclusion; and d) establishing, at the start
of the study, a one-month run-in period in order to identify and select participants with a
greater likelihood of compliance with the protocol and retention into the study (see
section 5 and 13).
C. Homogeneity of the interventions is difficult because it is based on three components:
diet, physical exercise, and behavior. For this reason we have developed a detailed
protocol for implementing the intervention and have established a committee for each
intervention component. We also conducted a staff training session at the beginning of
the study and will conduct annual follow-up sessions throughout.

COMMITTEES AND GOVERNANCE

The PREDIMED-PLUS Executive Committee includes the principal investigators
from all the participating centers (see Annex 1). It will provide scientific and strategic
orientation for decision-making and will be responsible for designing, implementing and
publishing the study’s protocol and guaranteeing the quality of its implementation and
management. It will determine its own guidelines and approve the criteria and guidelines
of the other committees within the study. It will convene at least twice a year to discuss
and report on the study’s progress.
The Steering Committee, made up of Jordi Salas Salvadó (Chair), Miguel Angel
Martínez-González (PI of the ERC-Advanced Research Grant), Ramón Estruch,
Montserrat Fitó, Emilio Ros, and Dolores Corella, is responsible for ensuring the quality
of the project and correcting any flaws or divergences that may be detected.

Data Safety and Monitoring Board

To ensure the smooth running of the trial and the safety of participants, an
Independent Data Safety and Monitoring Board has been set up. This Board is made
up of: Chairman, Meir J. Stampfer (Harvard School of Public Health); members Joan
Sabaté (Loma Linda University), Arne Astrup (Copenhagen University), Francisco
Fernandez-Avilés (Universidad Complutense of Madrid) and Xavier Pi-Sunyer (Columbia
University). The Board will convene at least once a year to review the implementation of
the protocol and monitor the trial’s progress. It will examine the competence of each
recruiting center, evaluate their compliance with the study’s objectives, and decide
whether they may continue in the trial.

54
In addition, a report will be mailed periodically by the PREDIMED-PLUS Steering
Committee to the Board members with relevant statistical analyses for judging on the
continuation of the PREDIMED-PLUS trial. Throughout the study, the Board members
can request any statistical analysis on a blinded or unblinded basis. The Board may
recommend termination of the trial at any time if an unacceptable incidence of adverse
events or significant differences in mortality between study groups are observed. The
Executive Committee of the PREDIMED-PLUS trial, however, will make the final
decision.

SOURCES OF FUNDING AND ADMINISTRATIVE ISSUES

This study was supported by the official funding agency for biomedical research of the
Spanish government, Instituto de Salud Carlos III (ISCIII) through the Fondo de
Investigación para la Salud (FIS) (grant to Jordi Salas-Salvadó) and CIBEROBN, which
is co-funded by the European Regional Development Fund, and by the European
Research Council (Advanced Research Grant 2013-2018; 340918) grant to Miguel Ángel
Martínez-Gonzalez.

Trial’s website
http://www.predimedplus.com

Contact’s name and address

Jordi Salas-Salvadó, MD, PhD. Human Nutrition Unit. Faculty of Medicine and Health
Sciences, Universitat Rovira i Virgili, C/ Sant Llorenç, 21, 43201 Reus (Spain).
Telephone number: +34 977759312; Fax number: +34 977759322. E-mail:
jordi.salas@urv.cat.
Prof. Miguel A. Martínez-González, MD, PhD, MPH, Department of Preventive
Medicine and Public Health, Facultad de Medicina–Clínica Universidad de Navarra,
Irunlarrea 1, 31008 Pamplona, Spain. Phone: +34 636 355 333, E-mail:
mamartinez@unav.es

PREDIMED-PLUS Registration
The PREDIMED--Plus trial was registered at the International Standard Randomized
Controlled Trial (ISRCT; http://www.isrctn.com/ISRCTN89898870) with number
89898870 and a registration date of 24 July 2014.

55
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ANNEX 1. SUB-STUDIES

1. Body composition
DEXA or computed tomography will be used to measure body composition at recruiting
centers wherein the necessary equipment and technology are available. Body
composition will be analyzed by General Electric Lunar DEXA scanner at the Rovira i
Virgili University, Universitat de les Illes Balears, Hospital Clinico de Barcelona, and the
Departments of Preventive Medicine and Nutrition in Navarra.

2. Other sub-studies
Depending on available funds, sub-studies will be conducted to evaluate gene
environment interactions, epigenetic factors such as DNA methylation, histone
modification and microRNA alterations, the composition and function of intestinal
microbiota by pyrosequencing, and the effect of the intervention on metabolomics,
transcriptomics and proteomics.

70
ANNEX 2. PARTICIPATING CENTERS

Recruitment centers

ID PI Email address Center

01 Enrique Gómez- egomezgracia@gmail.com Universidad de Málaga, Málaga
Gracia
02 José Lapetra jlapetra@ono.com Distrito Sanitario de Atención Primaria
de Sevilla, Sevilla
03 J. Alfredo Martínez jalfmtz@unav.es Universidad de Navarra - Nutrition,
Pamplona
04 María Adoración mariaadoracion.romaguera@ Hospital Son Espases, Palma
Romaguera;
Miquel Fiol ssib.es; miquelfiol@yahoo.es
05 Ramon Estruch restruch@clinic.ub.es Hospital Clínic-Medicina Interna,
Barcelona
06 Montserrat Fitó mfito@imim.es Institut Hospital del Mar
d’Investigacions Mèdiques, Barcelona
07 Jordi Salas-Salvadó jordi.salas@urv.cat Universitat Rovira i Virgili, Reus
08 Aurora Bueno abueno@ugr.es Universidad de Granada, Granada
09 Clotilde Vázquez cvazquezma@gmail.com Hospital Ramón y Cajal, Madrid
10 Miguel Ángel mamartinez@unav.es Universidad de Navarra -
Martínez-González Epidemiology, Pamplona
11 Fernando Aròs aborau@htxa.osakidetza.net Hospital Universitario Araba, Vitoria
12 Dolores Corella dolores.corella@uv.es Universidad de Valencia, Valencia
13 Lluis Serra-Majem lserra@dcc.ulpgc.es Universidad de las Palmas de Gran
Canaria, Las Palmas
14 Xavier Pintó xpinto@csub.scs.es Hospital de Bellvitge, L’Hospitalet de
Llobregat, Barcelona
15 José López Miranda jlopezmir@gmail.com Universidad de Córdoba, Córdoba
16 José Maria Ordovás Jose.Ordovas@tufts.edu IMDEA, Madrid
17 Pilar Matía Martín pilar.matia@gmail.com Hospital Clínico, Madrid
18 Francisco fjtinahones@hotmail.com Hospital de Málaga, Málaga
Tinahones
19 Josep Tur pep.tur@uib.es
Universitat de les Illes Balears, Palma
20 Josep Vidal JOVIDAL@clinic.ub.es Hospital Clínic – Endocrinología,
Barcelona

71
Support centers

ID PI Email address Center

A1 Emilio Ros eros@clinic.ub.es Hospital Clínic, Barcelona
A2 Rosa M. Lamuela- lamuela@ub.edu Universitat de Barcelona
Raventós
A3 Guillermo Sáez guillermo.Saez@uv.es Universitat de Valencia
A4 María del Puy mariapuy.portillo@ehu.es Universidad del País Vasco
Portillo
A5 Ascensión Marcos amarcos@ictan.csic.es CSIC, Madrid
A6 Cristina Botella botella@uji.es Universitat Jaume I de
Castellón
A7 Fernando ffernandez@bellvitgehospital.cat Hospital Universitari de
Fernández-Aranda Bellvitge

72
 EFFECT OF AN ENERGY-RESTRICTED
MEDITERRANEAN DIET, PHYSICAL ACTIVITY AND
BEHAVIORAL TREATMENT ON THE PRIMARY
PREVENTION OF CARDIOVASCULAR DISEASE

THE PREDIMED-PLUS TRIAL

RESEARCH PLAN

April 2018

IN THIS VERSION OF THE PROTOCOL THE AMMENDMENTS DONE FROM 2014

TO APRIL 2018 ARE INCLUDED

1
ABSTRACT
In the 21st century we are witnessing an alarming increase in overweight and
obesity. In Spain, over 60% of adults are overweight or obese and the prevalence of
adult abdominal obesity exceeds 35% (Gutiérrez-Fisac et al., 2012). The increase in
morbid obesity is especially worrying (Basterra-Gortari et al., 2011) because the
medium-to-long-term consequences for the risk of cardiovascular disease (CVD) and
other causes of death or illness can be catastrophic.
Observational studies have shown that all-cause mortality increases progressively
in parallel with adiposity and that the risk of cardiovascular mortality is especially high
(Berrington de González et al., 2010; GBD 2015 Obesity Collaborators). However, a meta-
analysis published in early 2013 (Flegal, 2013) raised considerable controversy by much
downgrading the consequences of overweight and moderate obesity. The results of this
meta-analysis could be explained by the existence of biases, such as unusual definitions
for categories of overweight, tobacco as a confounding factor, inverse causality, and the
exclusion of relevant studies (Tobias and Hu, 2013). Nevertheless, the controversy
persists and will probably do so well into the next few decades. Doubts such as these
will only be solved by randomized intervention trials (Hernán and Taubman, 2008). Thus,
clinical studies are needed to determine whether intentional weight loss reduces
cardiovascular mortality and CVD incidence.
In the PREDIMED-PLUS trial we will evaluate the safety and effectiveness of a
multifaceted intervention program for alleviating excessive cardiovascular morbidity and
mortality in overweight and obese individuals. The study’s aim is to determine the effect
on CVD morbidity and mortality of an intensive weight loss intervention program based
on an energy-restricted traditional Mediterranean diet, increased physical activity and
behavioral support in comparison with an intervention based on standard dietary advice
(energy-unrestricted Mediterranean diet) and traditional health care for CVD prevention.
We hypothesize that an intensive lifestyle intervention program aimed at weight loss and
based on the traditional Mediterranean diet is a sustainable long-term approach for
achieving weight loss in overweight and obese adults and that the lifestyle changes
achieved will have a beneficial effect on cardiovascular morbidity and mortality (Estruch
et al., 2013; Shai et al., 2008). This study may provide a useful tool for tackling excess
morbidity and mortality associated with overweight and obesity.

Objectives
The PREDIMED-PLUS trial evaluates the effect on primary CVD prevention of an
intensive intervention program comprising a 17-item energy-restricted Mediterranean

2
diet with specific weight-loss goals, physical activity promotion and behavioral support
(intervention group) in comparison to a control group using Mediterranen diet
recommendations (comprising a 14-item energy-unrestricted Mediterranean diet) but
without advice to increase physical activity or reduce energy intake (Schröder et al.,
2011) (control group). The diet assigned to the control group had previously been shown
to be effective for CVD prevention in the PREDIMED trial (Estruch et al., 2013). The
primary end-point is a composite of hard clinical cardiovascular events (myocardial
infarction, stroke or cardiovascular death) as defined in the PREDIMED trial (Martinez-
Gonzalez et al., 2012).

The main objective of PREDIMED-PLUS is to evaluate, in comparison with a control

group given non-intensive, energy-unrestricted dietary advice (also Mediterranean-
type), the effect of an intensive lifestyle intervention comprising an energy-restricted
Mediterranean diet, increased physical activity and behavioral support on:

1. The incidence of cardiovascular events (non-fatal myocardial infarction, non-fatal

stroke, or cardiovascular death).

2. Weight loss and long-term maintenance of weight-loss.

The secondary objectives are to determine whether an intensive weight-loss-

oriented lifestyle intervention program has a beneficial effect to reduce waist
circumference and the following overweight- and obesity-related conditions: acute
coronary syndromes with or without coronary revascularization, heart failure, atrial
fibrillation, peripheral artery disease, venous thrombosis, type-2 diabetes mellitus and its
complications, overall incidence of cancer, specific cancers in main cancer sites (breast,
colorectal, prostate, lung and stomach), osteoporotic fractures, gallstone disease,
symptomatic gout, neurodegenerative disorders (dementia and Parkinson’s disease),
unipolar depression and eating behavior disorders. We will also determine the effect of
the intervention on the following intermediate markers: nutrient intake and overall dietary
pattern, systolic and diastolic blood pressure, blood lipids, fasting glucose level, kidney
function, uric acid level, the percentage of individuals requiring anti-hypertensive, anti-
diabetic or lipid-lowering medication, C-reactive protein levels, hemoglobin A1C levels,
liver function, ECG traits, cognitive function, quality of life, and psychopathological
symptoms.

3
Methodology
We are conducting a randomized, multicenter field trial aimed at the primary
prevention of CVD in overweight or obese adults with metabolic syndrome using an
intensive intervention program based on an energy-restricted Mediterranean diet,
increased physical activity, and behavioral support and a control group given advice on
an ad libitum Mediterranean diet for the prevention of cardiovascular morbidity and
mortality in accordance with the PREDIMED trial. We stress to participants the
importance of attending medical visits and provide them with general written
recommendations on lifestyle for the management of the metabolic syndrome. This new
trial, the planning and design of which is outlined in this paper, is called PREDIMED-
PLUS.
There are 23 recruiting centers involved in this new multicenter trial including a total
of 6,874 participants, of whom 3,406 were assigned to the intensive intervention group
and 3,468 to the control group. Recruitment began in September 2013 and ended in
December 2016. The intervention will last at least 6 years and the median follow-up time
for the clinical endpoints is expected to be 8 years. The results of the trial, including
anthropometric changes and impact on major obesity-related disorders, are expected to
be highly applicable to public healthcare since they will provide a better prognosis for
overweight and obese adults. The results are also expected to be highly efficient since
they should provide a non-pharmacological approach to the prevention of the main cause
of mortality and one of the leading causes of loss of disability-adjusted life years (Lozano
et al., 2012).

4
BACKGROUND
The global overweight and obesity epidemic is increasing at an alarming rate.
Now a global public health crisis, it affects over 60% of the adult population. Between
1980 and 2008, worldwide obesity prevalence almost doubled (Finucane et al., 2011;
Malik et al., 2013; GBD 2015 Obesity Collaborators, 2017; González-Muniesa et al,
2017). In Spain, the prevalence of adult abdominal obesity is over 35% and over
60% of the adult population is overweight or obese (Gutiérrez-Fisac et al., 2012).
Moreover, there is a particularly worrying increase in morbid obesity (Basterra-Gortari et
al., 2011). The medium-to-long-term consequences of obesity on the risk of CVD and
death are devastating and have the capacity to both render the health system
unsustainable and curtail economic growth. Urgent priority must be given to finding
solutions based on the best scientific evidence available.
The link between obesity and mortality appears to diminish with age. However, if this
were true, it would be difficult to recommend weight loss for older populations. This notion
has been challenged by observational epidemiological studies that, after generational
and cohort confusion are adequately controlled, suggest the opposite, i.e., that with
advancing age the link between obesity and mortality becomes even stronger (Masters,
2013).
A meta-analysis of observational studies published in 2013 (Flegal, 2013) raised
controversy by considerably downgrading the consequences of overweight and
moderate obesity. However, as Tobias and Hu (2013) have argued, the conclusions of
Flegal’s meta-analysis can probably be explained by the existence of biases, such as
unusual definitions for categories of overweight, tobacco as a confounding factor, inverse
causality, and the exclusion of relevant studies. This issue will only be solved by
randomized clinical trials (Hernán and Taubman, 2008). However, the controversy is
likely to continue well into the next few decades until the results of clinical intervention
studies are available that overcome the limitations and inherent bias of the observational
designs that have evaluated the link between overweight or obesity and the incidence of
serious clinical events or mortality in initially healthy individuals. Observational studies
with a better control of bias have found that all-cause mortality increases progressively
as adiposity outside the normal weight range—measured by body mass index (BMI,
defined as weight in kilograms divided by the square of height in meters)—increases,
and that this risk is especially high for cardiovascular mortality (Berrington de
González et al., 2010; GBD 2015 Obesity Collaborators). An increase in body weight is
associated not only with higher mortality but also with greater morbidity due to CVD (Ni
Mhurchu et al., 2004; Song et al., 2004; Flint et al., 2010); greater risk of developing

5
some types of cancer (Lauby-Secretan B, et al 2016), diabetes and depression (Luppino
et al., 2010); and poorer cognitive function (Gunstad et al., 2010). Large-scale
randomized studies with robust designs are needed to obtain best-quality evidence.
Expert panels set up by the National Institutes of Health and the World Health
Organization recommend that overweight and obese adults with comorbid conditions
should lose 10% of their initial weight and that a lifestyle intervention program should be
the primary treatment (National Institute of Health, 1998; World Health Organization,
1998). Moreover, according to the American Dietetic Association, a weight-loss-oriented
intensive lifestyle intervention program should include an energy-restricted diet, physical
activity and behavioral therapy. The only randomized trial that has addressed the long-
term effect of an intensive weight-loss lifestyle program in obese adults on CVD and
mortality is the Look AHEAD study (Ryan, 2003). This trial, which comprised 5,145
participants (Rejeski et al., 2012), ended prematurely in October 2012 due to lack of
efficiency (Look AHEAD Research Group, 2013). The trial included only diabetic subjects
and used a low-fat diet (<30% of total energy intake with <10% from saturated fat). To
some extent this is opposite to the Mediterranean diet used in the PREDIMED trial
(Zazpe et al., 2008; Martínez-González et al, 2012; Estruch et al., 2013), which is rich in
vegetable fats such as healthful virgin olive oil and nuts. In recent years, scientific
associations have recommended low-fat diets that contribute less than 30% of energy in
the form of fat as the most suitable way to promote general health and weight loss. The
high energy density and high palatability of high-fat foods are feared to produce
potentially adverse effects on body weight and cardiovascular health (National Institute
of Health, 2000). However, the discontinuation of the Look AHEAD trial due to futility, the
inefficiency of the Women’s Health Initiative Dietary Modification Trial (Look Ahead
Research Group, 2013; Howard, 2006), and the favorable results of the PREDIMED
(Estruch et al., 2013) and DIRECT studies (Shai et al., 2008) provide powerful arguments
against approaches based on low-fat diets.
Although diets that recommend complex carbohydrates, a reduction in fat intake and
energy restriction to produce weight loss are generally accepted, there is no clear
evidence that dietary fat is associated with a greater increase in weight (Willett, 2001;
Nordmann et al., 2006; Larsen et al., 2010; Hu et al., 2012; Bueno et al., 2013). One
dietary paradigm that is different from the low-fat diet and that can be more useful for
developing and implementing programs aimed at achieving prolonged weight loss and
improving the metabolic alterations associated with overweight and obesity is the
traditional Mediterranean diet. This dietary pattern is rich in fat from vegetable sources
(virgin olive oil and nuts) and includes an abundance of minimally processed plant foods

6
(vegetables, fruits, whole grains and legumes), low consumption of meat (especially red
and processed meats), moderate consumption of fish and wine (which is usually
consumed with meals) and frugal meals. The high fat contents of the traditional
Mediterranean diet make the diet more palatable and therefore more acceptable and
easily sustainable in the long term.
In its 2010 edition, the Dietary Guidelines for Americans recognized the traditional
Mediterranean diet, together with the DASH diet, as a healthy diet for the prevention of
CVD, although when the recommendation was made, randomized clinical trials with
regard to the primary prevention of major clinical events as the main outcome had not
yet been conducted. This was confirmed by the results of the PREDIMED trial on the
primary prevention of cardiovascular disease conducted in Spain between 2003 and
2010 (Estruch et al., 2013).
With regard to the weight-loss properties of the traditional Mediterranean diet, in a
meta-analysis of randomized trials, allocation to a Mediterranean diet in comparison with
control diets showed a small but significant effect on body weight reduction (mean
differences: -1.75 kg, CI 95%: -2.86 a -0.64 kg). This effect was doubled when the
Mediterranean diet was energy-restricted (Esposito et al., 2011). Subsequently the
PREDIMED trial showed similar advantages for the prevention of weight gain in a
considerably longer-term follow-up and with a higher sample size (Estruch et al, 2016).
Several meta-analyses of observational studies (Sofi et al., 2010; Sofi et al, 2014;
Martínez-González et al, 2017; Dinu et al, 2018) found that greater adherence to the
Mediterranean diet was associated with significant reductions in total mortality,
cardiovascular mortality, mortality due to cancer, the incidence of non-fatal
cardiovascular events, and the risk of neurodegenerative illnesses. A subsequent update
of the meta-analysis of the Mediterranean diet and CVD reported a 13% relative
reduction in risk for every two-point increase in adherence to the Mediterranean diet
(scale 0–9) after identifying and treating sources of heterogeneity (Martínez-Gonzalez
and Bes-Rastrollo, 2014).
The PREDIMED (PREvención con DIeta MEDiterránea) trial, which included
7,447 participants over an average of five years, was the largest nutritional intervention
trial ever conducted in Europe. PREDIMED showed that, in comparison with advice on
a low-fat diet, a high-fat Mediterranean diet supplemented with extra-virgin olive oil or
mixed nuts implemented in a setting of primary cardiovascular prevention resulted in a
30% reduction in CVD events after intervention for a median of 4.8 years (Estruch et
al., 2013). PREDIMED is recognized worldwide as a landmark study that marks a turning
point in the prevention of chronic diseases. The effective reduction in cardiovascular

7
events when the Mediterranean diet was used in a randomized trial provides the best-
possible scientific evidence for preventing CVD, the main cause of death in the world.
We should also point out that the PREDIMED diets were ad libitum, increased physical
activity was not promoted, and no counsel to lose weight was given.
It has been postulated that the link between adherence to the traditional
Mediterranean diet and the risk of CVD can be mediated by several mechanisms,
including a reduction in low degree inflammation (Chrysohoou et al., 2004; Esposito et
al., 2004; Mena et al., 2009; Camargo et al., 2011; Urpi-Sarda et al., 2012; Meneses et
al., 2012), higher levels of adiponectin (Detopoulou et al., 2010; Razquin et al., 2010),
lower coagulability (Chrysohoou et al., 2004; Pérez-Jiménez et al., 2006; Pérez-Jiménez
et al., 2002), improved endothelial function (Esposito et al., 2004; Ruano et al., 2005;
Fuentes et al., 2008), lower oxidative stress (Dai et al., 2008; Chrysohoou et al., 2011;
Razquin et al., 2009), a lower concentration of atherogenic lipoproteins (Jones et al.,
2012), lower levels of oxidized LDL particles (Fito et al., 2007), and a lower uptake of
oxidized LDL by macrophages (Moreno et al., 2008). Moreover, the two foods
supplemented in PREDIMED (extra-virgin olive oil and nuts) also have beneficial
biological properties. Extra-virgin olive oil has a healthy fatty acid profile and contains
numerous bioactive phenolic compounds (Pérez-Jiménez et al., 2006; Covas et al.,
2009; López-Miranda et al., 2010). The phenolic compounds of olive oil have anti-
inflammatory properties (Fito et al., 2008), beneficially impact he lipid profile (Benkhalti
et al., 2002; Covas et al., 2006), improve oxidative stress markers (Covas et al., 2006),
have a platelet antiagregant effect (de Roos et al., 2011; Fito et al., 2008), and stimulate
mitochondrial biogenesis (Zhu et al., 2010). Nuts also have a healthy fatty acid profile,
based on mono- and polyunsaturated fatty acids, and contain minerals, vitamins and
other antioxidant bioactives, essential amino acids, fiber, and phytosterols (Ros, 2009).
The consumption of nuts has been associated with lower levels of total cholesterol, LDL
and non-HDL cholesterol, and apolipoprotein B-100 (Li et al., 2009; Sabaté et al., 2010),
and lower inflammation (Jiang et al., 2006). Nuts also have an antioxidant effect, benefit
heart rate and improve platelet aggregation and endothelial function (Ros, 2009;
Defilippis et al., 2010). All of these mechanisms explain the antiatherogenic effect of a
Mediterranean diet that is rich in nuts and extra-virgin olive oil. In fact, in the PREDIMED
trial a strong protective effect against peripheral artery disease was observed (Ruiz-
Canela et al., 2014). In a sub-study of the PREDIMED trial we also observed that both a
Mediterranean diet enriched with nuts and a Mediterranean diet enriched with olive oil
reduced the incidence of type-2 diabetes by 48% (Salas-Salvadó et al., 2011). When we
analyzed this association among all study’s participants, we also found that the

8
Mediterranean diet had a significant protective effect against diabetes (Salas- Salvadó
et al., 2014).
Though PREDIMED study, was not a weight loss trial, the provision of abundant fat-
rich foods from natural vegetable origin (extra-virgin olive oil and nuts) did not conduct
to weight gain. There is still insufficient experimental evidence to support the
hypothesis that intentional weight loss via a healthy diet and favorable lifestyle changes
reduces mortality or the incidence of CVD in the long term. Specifically, the impact of
weight loss on the risk of CVD within the framework of a Mediterranean dietary pattern
has not yet been tested in a sufficiently large randomized clinical trial (Malik and Hu,
2007). In light of the obesity epidemic, we propose to conduct a new trial, PREDIMED-
PLUS, which will go beyond the achievements of the PREDIMED trial in order to tackle
more specifically the problems of overweight and obesity. Our proposed strategy has
positive effects for weight loss (based on the loss of fat mass) and long-term weight-loss
maintenance (Shai et al., 2008; Beunza et al., 2010; Romaguera et al., 2010; Estruch et
al 2016). Even more interestingly, this research may demonstrate that a multifaceted
lifestyle intervention program (dietary pattern + weight loss + physical activity +
behavioral support) can be an even more effective means of reducing the cardiovascular
risk associated with overweight and obesity than a non-energy-restricted traditional
Mediterranean diet. We expect our contribution via the PREDIMED-PLUS trial to reveal
synergies between the effects of an intensive weight-loss intervention program (with
energy restriction, physical activity and behavioral support) and the beneficial effects
of greater adherence to a high-quality diet (the Mediterranean diet) on the incidence of
CVD.
Blood and urine samples will be collected and stored at the beginning and
throughout the trial. Later analyses of molecular/biochemical biomarkers within the
framework of genetic, epigenetic, transcriptomic, metabolomic and proteomic studies
might help to determine the benefits of the intervention and the underlying mechanisms.

HYPOTHESIS
An intensive lifestyle intervention program based on an energy-restricted
traditional Mediterranean diet, increased physical activity and behavioral support is a
sustainable approach that leads to long-term weight loss in overweight and obese adults
with metabolic syndrome in such a way that the changes in lifestyle achieved will have
long-term benefits on the incidence of CVD.

9
 In comparison with a control intervention that provides advice on the Mediterranean
diet but does not restrict calorie intake and does not promote physical activity, an
intensive lifestyle intervention based on an energy-restricted traditional Mediterranean
diet, promotion of physical activity, and behavioral support (Intervention group) in
overweight or obese individuals with metabolic syndrome will:
1. Reduce the risk of cardiovascular events;
2. Achieve a greater reduction in body weight and lead to better long-term weight-loss
maintenance;

OBJECTIVES
Our long-term objective is to provide effective treatment for reducing excessive
cardiovascular morbidity and mortality in overweight or obese adults, irrespective of
whether the participants are diabetic at the beginning of the study. To achieve this, we
will compare the effects on rates of cardiovascular disease of an intensive lifestyle and
weight loss intervention program based on the traditional Mediterranean diet and
including increased physical activity, energy restriction and behavioral support
(intervention group) with that of a non-intensive intervention program that provides both
education on the traditional Mediterranean diet for the prevention of CVD in
accordance with the principles outlined in the PREDIMED trial and usual care by
primary healthcare professionals (control group). The importance of attending visits to
healthcare professionals will be stressed and general recommendations on management
of the metabolic syndrome will be provided.

Main specific objectives

To evaluate the effect of an intensive weight-loss-oriented lifestyle intervention program
based on a traditional Mediterranean diet with energy restriction, increased
physical activity and behavioral therapy on:
1. The incidence of CVD (non-fatal myocardial infarction, non-fatal stroke and
cardiovascular death);
2. Weight loss and long-term weight-loss maintenance;

Secondary specific objectives

This intensive intervention program is likely to result in reduction of waist
circumference and acute coronary syndromes, coronary revascularization, total
mortality, heart failure, peripheral artery disease, venous thrombosis, atrial fibrillation,
type-2 diabetes and its complications, total cancer, cancer in main cancer sites (breast,

10
prostate, colorectal, lung and stomach), gallstone diseases, symptomatic gout,
neurodegenerative disorders (dementia and Parkinson’s disease), unipolar depression,
osteoporotic fractures, and eating behavior disorders.
We will also address the effect of the intervention on the following intermediate
outcomes: nutrient intake and overall dietary pattern, systolic and diastolic blood
pressure, serum lipid concentrations, fasting glucose, glycated hemoglobin and uric acid,
kidney function, liver function, C-reactive protein, anti-hypertensive, anti-diabetic and
lipid-lowering medication needs, ECG traits, cognitive function, quality of life, and
psychopathological scales.
We will also store plasma, serum, peripheral cells and urine samples to evaluate
other hypotheses in the future, depending on availability of additional funding.

METHODOLOGY
1. Summary
We are conducting a parallel-group, multicenter, randomized, primary prevention
trial in adult men aged 55–75 and adult women aged 60–75 with a BMI ≥27 and <40
kg/m2 who meet at least three criteria for the metabolic syndrome. In the Caucasian
population, cardiovascular risk is considered to be increased if waist circumference is ≥
80 cm in women and ≥ 94 cm in men. In the South American population, the value is the
same for women but for men risk is considered to increased if waist circumference is ≥
90 cm (Alberti et al., 2009). Diabetic participants comprise 27% of the final sample. This
latter component and the use of the traditional Mediterranean diet substantially
distinguish the PREDIMED-PLUS trial from the Look AHEAD trial conducted in the
United States (Ryan et al., 2003; Gregg et al., 2012; Look AHEAD Research Group,
2013), which was recently discontinued due to lack of efficiency. Participants were
divided proportionally at random into two groups: a control group and an intensive
lifestyle intervention group. Intervention will be maintained for 6 years and average
follow-up time for clinical events will be 8 years.
The control group receives usual healthcare from primary care medical
professionals, all the written material, instructions on following the traditional
Mediterranean diet—which was used in the PREDIMED study and has been shown to
have benefits for the prevention of cardiovascular morbidity and mortality—and general
lifestyle recommendations for managing the metabolic syndrome. Every six months, the
control group participants are also invited to participate in group sessions led by the team
of PREDIMED-PLUS dietitians, wherein they receive a free supply of virgin olive oil (6

11
liters every 6 months) and nuts (750 g every 6 months) in order to promote the
Mediterranean diet and encourage compliance with the trial.
Participants in the intensive lifestyle intervention group are prescribed a
traditional Mediterranean diet but in this case it is energy-restricted. Dietary
intervention is associated with increased physical activity and behavioral therapy
programs. It has specific weight-loss objectives and includes self-monitoring and
frequent follow-up throughout the study. Participants in this group take part in individual
interview sessions and motivational group sessions three times per month during the first
year of the intervention and twice per month thereafter. They are provided free extra-
virgin olive oil (one liter per month) and nuts (125 g per month of the total recommended
amount of 500g) . Initially, we decided to provide 500 g per month at no cost of nuts to
each participant during the intervention in order to reinforce adherence to the
Mediterranean diet in both arms of the trial. However, due to lack of economic resources,
only participants included in the pilot study recruited in 2 vanguard centers up to July
2014 received 250 g of free nuts (125g of pistachios and 125g of almonds per month)
along with the recommendation to consume a total monthly amount of 500g.
Subsequently, all trial participants received during the follow-up 125g of almonds per
month out of the total recommended amount of 500g. The participants’ degree of
compliance with the intervention is monitored periodically so that the intervention can be
adjusted if necessary.
For the intensive intervention group, the specific weight-loss objectives are to
achieve an average reduction in baseline body weight of over 8% and an average
reduction in waist circumference of over 5% in the first six months and to maintain
these figures over an additional period of seven and a half years. The final objective is
to obtain a between-group average absolute difference in weight loss and waist
circumference reduction of over 5%.
Primary final outcomes include: a) non-fatal myocardial infarction, b) non-fatal
stroke, and c) cardiovascular death. Other primary objective will be weight loss (and
weight-loss maintenance). The trial protocol will be registered at ClinicalTrials.gov

The decision to recommended 500 g of mixed nuts per month was based on the results of epidemiological
and clinical studies. For example, the SUN (Seguimiento Universidad de Navarra) study found that
individuals who consumed nuts two or three times per week (400 g/month) had a significantly lower risk of
weight gain and metabolic syndrome than those who rarely ate them or never did (Fernández-Montero et
al., 2012; Bes-Rastrollo et al., 2007). A Mediterranean-type diet with moderate fat intake containing 25 g
per day (750 g/month) of peanuts or other types of nuts was found to be associated with better adherence
to intervention and greater weight loss than a low-fat diet (McManus et al., 2001). The decision was also
based on associations observed between baseline nut consumption and mortality in the PREDIMED trial
(Guasch-Ferre et al., 2013) and in American cohorts of nurses and healthcare professionals (Bao et al.,
2013).

12
(National Institutes of Health) and comply with the CONSORT guidelines for the
dissemination of results (Moher et al., 2001).

2. Research team
The trial comprise 6,874 participants, 3,406 were assigned to the intensive
intervention group and 3,468 to the control group. Recruitment of participants began in
September 2013 and ended in December 2017. The 2 vanguard centers were Navarra-
Epidemiology (starting the recruitment in September 2013 and the randomization in
October 2013) and Reus (starting the recruitment in November 2013, and the
randomization in January 2014). Training of dietitians for the other recruiting centers took
place in December 2013. Initially, our recruitment objective with 20 centers was that they
each recruiting an average of 300 participants. But, In order to increase the possibility of
meeting target numbers of participants recruited by December 2017, in November 2014,
the Steering Committee accepted the inclusion of three additional recruiting centers
[Universidad Miguel Hernández, Sant Joan d'Alacant, Alicante (IP: Jesús Vioque);
Universidad de Jaén, Jaén (IP: Miguel Delgado Rodríguez) y; Universidad de León, León
(IP: Vicente Martín)]. The final sample included 6,874 participants distributed in 23
centers.
To launch the trial, we have set up a team of leading researchers with experience in
diet and lifestyle interventions and productive and well-documented collaborative
research careers in nutrition, evaluation of physical activity, internal medicine,
cardiology, endocrinology, primary health care, epidemiology, and basic sciences.
The prior experience gained by the 11 PREDIMED recruiting centers is one of our
most valuable assets for the PREDIMED-PLUS trial. Also, by incorporating other centers
of scientific excellence with proven experience in nutritional intervention clinical trials
(some of which also belong to the background research network of the PREDIMED trial,
CIBEROBN), the correct development of the PREDIMED-PLUS trial will be guaranteed
and feasibility will be improved.
At the same time, subprojects will be devised so that all participating groups can
develop their own specific nutritional research activities. This will also serve to further
interest in the project and enhance scientific output.

3. Preliminary studies: the PREDIMED trial

In this section we present a summary of the methodology and key findings of the
PREDIMED trial (Estruch et al., 2006; Zazpe et al., 2008; Martínez-González et al., 2012;
Estruch et al., 2013), which was conducted in the context of CIBEROBN and the

13
PREDIMED network (RD06/0045). PREDIMED was a multicenter, parallel group, trial
with three intervention groups (see www.predimed.es). In 2006, the results of a pilot
study were published that evaluated the effects at three months of the three interventions
on classical and emergent cardiovascular risk factors in the first 772 participants (Estruch
et al., 2006). The design and methods of the PREDIMED trial have been described
elsewhere (Martínez-González et al., 2012). Participants in the study were men aged 55-
80 and women aged 60-80 without CVD at the beginning of the study but with a high risk
of CVD due to the presentation of type 2 diabetes or at least three of the following six
cardiovascular risk factors: smoking, high blood pressure, high LDL cholesterol level, low
HDL cholesterol level, overweight or obesity, and family history of early coronary heart
disease.
Candidates for the trial were recruited at primary care health centers. Eighty-nine
per cent of those invited to participate agreed to do so and signed the corresponding
informed consent form. The final sample size for the trial was 7,447 participants. The
protocol was approved by the ethics research committees of all study centers and
registered at the Clinical Trials Register in London (ISRCTN35739639).
The participants were randomly assigned in a 1:1:1 ratio to one of the following three
dietary intervention groups: 1) an energy-unrestricted Mediterranean diet
supplemented with extra-virgin olive oil; 2) an energy-unrestricted Mediterranean diet
supplemented with nuts; or 3) an energy-unrestricted control diet with advice on how to
follow a low-fat diet.
At the beginning of the study and quarterly thereafter, dietitians conducted individual
and group dietary-training sessions (separately for each group) with a maximum of 20
participants per group (Zazpe et al., 2008). At each session of the Mediterranean diet
groups, a 14-item questionnaire (Martínez-González et al., 2004; Schroeder et al., 2011)
was used to assess participants’ adherence to the Mediterranean diet. At each session
of the control group, a 9-item questionnaire was used to assess participants’ adherence
to the control diet. In this way the diets could be personalized and appropriate dietary
changes could be negotiated individually.
Participants in the two Mediterranean-diet groups received either extra-virgin
olive oil (1 liter per week for the participant and his or her family) or 30 g of mixed nuts
per day (15 g of walnuts, 7.5 g of hazelnuts, and 7.5 g of almonds) at no cost and in
accordance with their randomly chosen group, while those in the control group received
small non-food gifts throughout the trial. At no point during the intervention was calorie
restriction advised or increased physical activity encouraged.

14
 Information on the primary end points (cardiovascular death, non-fatal stroke, or
acute non-fatal myocardial infarction) was obtained through continuous contact with the
participants, contact with primary healthcare physicians, ad hoc annual reviews of
medical records conducted by a medical team at each center, and annual consultation
of the National Death Index. The data were analyzed on an intention-to-treat basis.
Participants were followed for a median of 4.8 years.
Participants in the two Mediterranean diet groups increased their adherence to the
diet, as reflected in an average increase of 2 points on the 14-point dietary-screening
questionnaire throughout the duration of the study. These participants also had higher
average scores than those in the control group on all items except red and processed
meats and sugary soda drinks, which were discouraged for all three intervention groups.
After a median follow-up of 4.8 years, 288 participants suffered a major
cardiovascular event. In comparison with the control group, the multivariable-adjusted
hazard ratios were 0.70 (95% confidence interval [CI], 0.54 to 0.92) for the group
assigned to the Mediterranean diet with extra-virgin olive oil and 0.72 (95% CI, 0.54 to
0.96) for the group assigned to the Mediterranean diet with nuts.

FIGURE 1. Cumulative incidence of major cardiovascular events (cardiovascular

death, non-fatal myocardial infarction, and non-fatal stroke) by randomly assigned
group (n=7,447).

15
4. Proposed trial (PREDIMED-PLUS) and eligibility criteria
Candidates for the PREDIMED-PLUS trial are adults aged 55-75 for men and 60-75
for women with a body mass index ≥27 and <40 kg/m2 who meet at least three criteria
for the metabolic syndrome (Alberti et al., 2009). These criteria must be taken into
consideration in view of recent evidence of the beneficial role of the Mediterranean diet
on the metabolic syndrome (Kastorini et al., 2011; Salas-Salvadó et al., 2008), insulin
resistance and diabetes (Salas-Salvadó, 2014), especially when accompanied by a
program of physical activity for endurance (Fernández et al., 2012). Approximately, 50%
of the study population is made of women and diabetic participants do not exceed 27%
of the total cohort. Individuals who participated in the PREDIMED trial was not eligible to
participate in PREDIMED-PLUS.

4.1. Exclusion criteria:

• Illiteracy or inability/unwillingness to give written informed consent or communicate with
study staff.
• Institutionalization (the participant is a permanent or long-stay resident in a care home).
• Documented history of previous CVD, including: angina; myocardial infarction; coronary
revascularization procedures; stroke (ischemic or hemorrhagic, including transient
ischemic attacks); symptomatic peripheral artery disease that required surgery or was
diagnosed with vascular imaging techniques; ventricular arrhythmia; uncontrolled
atrial fibrillation; congestive heart failure (New York Heart Association Class III or IV);
hypertrophic myocardiopathy; and history of aortic aneurism >=5.5 cm in diameter or
aortic aneurism surgery.
• Active malignant cancer or history of malignancy within the last 5 years (except non-
melanoma skin cancer).
• Inability to follow the recommended diet (for religious reasons, swallowing disorders,
etc.) or to carry out physical activity.
• A low predicted likelihood to change dietary habits according to the Prochaska and
DiClemente Stages of Change Model (Nigg et al., 1999).
• Inability to follow the scheduled intervention visits (institutionalization, lack of autonomy,
inability to walk, lack of stable address, travel plans, etc.).
• Inclusion in another program that provides advice on weight loss (> 5 kg) in the six
months before the selection visit.
• History of surgical procedures for weight loss or intention to undergo bariatric surgery
in the next 12 months.
• History of small or large bowel resection.

16
• History of inflammatory bowel disease.
• Obesity of known endocrine origin (except for treated hypothyroidism).
• Food allergy to any component of the Mediterranean diet.
• Immunodeficiency or HIV-positive status.
• Cirrhosis or liver failure.
• Serious psychiatric disorders: schizophrenia, bipolar disorder, eating disorders, or
depression with hospitalization within the last 6 months.
• Any severe co-morbidity condition with less than 24 months’ life expectancy.
• Alcohol abuse or addiction (or total daily alcohol intake >50 g) or drug abuse within the
past 6 months.
• History of major organ transplantation.
• Concurrent therapy with immunosuppressive drugs or cytotoxic agents.
• Current treatment with systemic corticosteroids.
• Current use of weight loss medication.
• Concurrent participation in another randomized clinical trial.
• Patients with an acute infection or inflammation (e.g. pneumonia) will be allowed to
participate in the study 3 months after resolution of their condition.
• Any other condition that may interfere with adherence to the study protocol.

5. Recruitment and retention strategies

Medical doctors from primary care centers associated with the recruiting centers
recruited the participants. The mission of the primary care physicians was to ensure a
high recruitment rate and an almost 100% diligence in the revision of medical records
and collection of clinical information on events during follow-up. As the physicians
involved in the recruitment process are also responsible for the participants’ medical
care, no potential ethical conflict regarding confidentiality exists when identifying suitable
candidates or reviewing medical records. Participants’ eligibility criteria and demographic
data were collected from the medical records at the primary care centers, which are
entirely computer-based. This was done at a pre-screening evaluation stage before the
potential participant is contacted. Candidates were interviewed briefly by telephone,
informed about the study, and invited to attend a screening visit at the recruiting center.

In this first formal visit (first screening visit), the candidates were explained the
purpose and characteristics of the study and, if they agreed to participate, they were
asked to sign a written informed consent. Our experience with the PREDIMED trial
showed that over 95% of eligible candidates approached in this way agreed to participate

17
(Martínez-González et al., 2012; Estruch et al., 2013). Also, in the PREDIMED trial,
which also included a long-term lifestyle intervention program (median follow-up time of
4.8 years), overall retention rate was above 90% (Estruch et al., 2013).

6. Informed consent/Ethics Committee

The institutional review boards of all the recruiting centers approved the study
protocol. As described and detailed below, all participants signed informed written
consent forms.

7. Launch of the trial

The proposed calendar was as follows:
• Between August 2013 and January 2014, the dietitians and nursing staff were hired
and trained to deliver the trial’s protocol.
• Between August 2013 and May 2014, eligible candidates began to be called,
interviewed and invited to participate in the trial.
• Next, the first evaluation and intervention visits took place with randomly distributed
selected participants.
• The recruitment period will end on December 2016.

7.1. The aim of the initial stage of the trial (telephone calls and interviews) was to
evaluate the willingness of each candidate to participate in the study, comply with the
proposed intervention, and lose weight. In addition, they were thoroughly screened in
order to ensure that the eligibility criteria were met and evaluated the probability that
they:
a) attend the scheduled sessions,
b) complete the protocol’s assessment tools, i.e., the self-monitoring and recording of
lifestyle and food habits and, most importantly,
c) change their dietary habits in accordance with the Stages-of-Change model (Nigg
et al., 1999), as occurred in the PREDIMED trial.

7.2. The run-in period (for evaluation prior to randomization) lasts four weeks. It
comprised an initial screening visit, a phone call at 2 weeks, and a final evaluation visit.
7.2.1. The first screening visit (45-60 min) comprised:
a) administration of a questionnaire on inclusion and exclusion criteria. Candidates
who were deemed eligible to participate in the trial continue to the next stage.

18
 b) explanation of the study, distribution of the study information sheet, and
completion of the informed consent forms (these are essential for inclusion in the
trial). Eligible candidates were asked to sign two informed consent forms: one for
participation and analysis of general variables and one for the collection of DNA for
genetic analyses. All procedures and anticipated time commitment were explained in
detail. Candidates were also told that, if they did not satisfy the eligibility criteria, they
would be excluded from the study. The informed consent form includes a statement
allowing researchers to review the participants’ medical records throughout the trial
at both the primary care centers and reference hospitals in order to ascertain the
occurrence of any events.
c) performance of ECG and recording of height, weight, waist circumference and
blood pressure.
d) distribution of a leaflet containing general recommendations on managing the
metabolic syndrome.
e) Distribution of a 3-day food record questionnaire (2 working days and 1 weekend
day), a leisure-time physical activity questionnaire, and a self-measurement chart in
which participants self-record their weight, waist circumference and hip circumference
(participants are given a tape measure). Dietitians gave instructions on how and when
to complete the food record and physical activity questionnaires and how to record
their weight, waist and hip measurements (once a week during the trial).
f) Distribution of the clinical psychopathological questionnaires (Beck Depression
Inventory (BDI-II), multidimensional scale of weight locus of control, eating disorders
diagnostic criteria, and quality of life scale (SF-36)) to be completed at home (see
below).
Participants were asked to return their completed questionnaires at the third
screening visit (see below).
7.2.2. Second screening visit. After 2 weeks, participants received a telephone call to
assess their change in weight and remind them to bring to the next screening visit
their completed food record, and physical activity questionnaires, self-measurement
chart, psychopathological questionnaires, and quality of life scale.
7.2.3. Third screening visit. This evaluation visit on completion of the four-week run-in
period (30 min) included:
a) Collection by the dietitian of the participants’ food record and physical activity
questionnaires, self-measurement charts, psychopathological questionnaires, and
quality of life scales.
b) Measurement by the dietitian of the participants’ weight and hip circumferences.

19
 c) Administration and completion of a 143-item food-frequency questionnaire and 5
cognitive-neuropsychological tests, which, unlike the clinical psychopathological
questionnaires, were completed in the presence of PREDIMED-PLUS personnel.
These 5 tests are: the Mini-Mental State examination, the phonological verbal fluency
test, the reverse digits test, the trail making test, and the clock test (see below).
d) Explanation to participants that they would be informed by telephone if they had
been selected to participate in the trial.
e) Explanation of night-time fasting for in situ extraction of blood sample and first
morning urine sample, and of basal evaluation immediately after randomization for
candidates who were chosen to participate in the trial.

Only candidates who satisfied the following four criteria were selected and randomly
assigned to one of the two intervention groups:
1) Full attendance at the two previous sessions, at the scheduled times and having
answered the telephone call;
2) Correct completion of the clinical psychopathological questionnaires (Beck
Depression Inventory (BDI-II), multidimensional scale of weight locus of control, eating
disorders diagnostic criteria, and quality of life scale (SF-36);
3) Correct completion of the food record and physical activity questionnaires;
4) Correct self-recording of at least three weight measurements and three waist-
circumference measurements.
Note, that initially the lost of >1.5 kg during the run-in period was one selection criteria.
However, In January 2014, the Steering Committee following the advice of the Data
Safety and Monitoring Board decided to amend the Protocol and omit the 1.5 kg weight
loss criterion to be achieved during the run-in period. Such change only affected the first
70 participants who were eligible and randomized in 2 vanguard centers.

The lag time between completion of the run-in phase and the beginning of the
intervention ranges from one week to one month.
Exceptionally, depending on the availability of the candidate to attend the Run-in S3 visit,
and considering that the 4 mentioned criteria are met, the candidates can be randomized
to one of the two intervention groups after completing Run-in S2 visit face-to-face.

8. Initial screening, follow-up visits, and evaluations

Table 1 shows the main data collection measurements and activities by visit.

20
TABLE 1. The following data are collected per visit in the PREDIMED-PLUS trial.
RUN-IN PERIOD
S1 S2 S3 Baseline M-6 Y-1 Y-2 Y-3 Y-4 Y-5 Y-6 Y-7 Y-8
1. ELIGIBILITY QUESTIONNAIRE X
2. 3-DAY FOOD REGISTER e c
3. ANTHROPOMETRIC MEASUREMENTS* X X X X X X X X X X X X
4. GENERAL QUESTIONNAIRE X
5. 143-ITEM FFQ X X X X X X X X X X
6. MEDITERRANEAN DIET QUESTIONNAIRE (17/14-Items)** X X X X X X X X X X
7. PHYSICAL ACTIVITY QUESTIONNAIRE‡ e† c† X X X X X X X X X X
8. CHAIR TEST (Physical activity evaluation) X X X X X X X X X X
9. ACCELEROMETERS e X X X X X X X X X X
10. FOLLOW-UP QUESTIONNAIRE X X X X X X X X X
11. ELECTROCARDIOGRAM X X X X X X X X X X
12. BLOOD PRESSURE MEASUREMENT X X X X X X X X X X X X
13. BLOOD SAMPLE COLLECTION X X X X X X X
14. MORNING SPOT URINE COLLECTION X X X X X X X
15. NAIL COLLECTION X X X X X X
16. COGNITIVE-NEUROPSYCHOLOGICAL TESTS≠ X X X X X
17. PSYCHOPATHOLOGICAL QUESTIONNAIRES€ e X X X X X X X X X
18. QUALITY OF LIFE QUESTIONNAIRES≈ e X X X X X
S: Screening visit; FFQ: Food-frequency questionnaire; M: Month; e: Delivery; c: Collection.
* Anthropometric measurements included: weight, height, waist circumference and hip circumference.
‡
Short version of the Minnesota leisure time physical activity questionnaire; PAR-Q, RAPA (RAPA1 and RAPA2) questionnaires; and the NHS sedentary lifestyle questionnaire
†
Long version of the Minnesota leisure time physical activity questionnaire.
**Short questionnaires on adherence to the Mediterranean Diet. The control group uses the same 14-item questionnaire that was used in the PREDIMED trial (Schroeder et al.,
2011). The intervention group uses the 17-item energy-restricted Mediterranean diet questionnaire (see below).
≠Mini-Mental Status Examination, clock test, phonological verbal fluency test (animals + P), the reverse series of digits test (WAIS-III), and the trail making test.

€Beck Depression Inventory (BDI-II), multidimensional scale of weight locus of control, eating disorders diagnostic criteria, and SF-36 quality of life scale.

21
Eligibility: Eligibility for inclusion in the trial was assessed at the beginning of the study.
Selected participants must satisfied all the eligibility criteria. Exclusion criteria were also
verified.
Anthropometric measurements: Weight and waist circumference are recorded at each
visit, with participants in light clothing and without shoes or accessories, using a high-
quality electronic scale that is calibrated every 3 months with a unit of known mass.
Height is measured at study entrance with a stadiometer. Waist circumference is
measured midway between the lowest rib and the iliac crest. Hip circumference is
measured at the widest part at the baseline visit and on a yearly basis.
General information: Information on medical history, family history and use of medication
is collected at the baseline visit by means of the general questionnaire, using the same
protocol as in the PREDIMED trial (See supplementary file in Estruch R et al., New Engl
J Med 2013).
Evaluation of food habits and dietary intake: The previously validated 143-item food-
frequency questionnaire is administered at the third screening visit and at each annual
follow-up visit to evaluate the diet of each participant (Fernández-Ballart et al., 2010). In
addition, the 17-item energy-restricted Mediterranean diet questionnaire (see below) is
completed at each visit. This questionnaire, which includes several changes with respect
to a previously validated tool (Schroder et al., 2011), is used both to assess participants’
compliance with the intervention and to guide the individual motivational interviews
during follow-up. The control group, on the other hand, is administered the same 14-item
questionnaire that was used for the PREDIMED trial (Schröder et al., 2011).
Physical activity: Except for the first screening visit (when participants complete the long
Minnesota leisure time physical activity questionnaire), at the beginning of the study,
after 6 months, and during the follow-up visits, participants will complete a short version
of a previously validated physical activity questionnaire (Elosua et al., 1994; Elosua et
al., 2000). Also at these latter visits (baseline, after 6 months, and during the follow-up
visits), participants will perform the chair test (30 seconds) in order to evaluate their
physical fitness and complete the following questionnaires: the PAR-Q (Physical Activity
Readiness Questionnaire), the RAPA (RAPA1 and RAPA2) (Rapid Assessment of
Physical Activity), and the NHS (Nurses’ Health Study) sedentary lifestyle questionnaire.
All these questionnaires are described in the PREDIMED-PLUS website:
http://www.PREDIMEDPLUS.COM. Each participant randomly assigned to the
intervention group is also provided with a pedometer (Yamax SW200 Digi-Walker) to
self-monitor the number of steps walked per day. GENEActiv accelerometers are
provided as well to a subset of participants (50% of participants in the intensive

22
intervention group and 20% of those in the control group) in order to quantify physical
activity at baseline, 6 months, 1 year, and each year thereafter. In accordance with an
evaluation based on physical status, recommendations on aerobic physical activities and
strength training are progressively made and activities to improve balance and flexibility
encouraged on completion of physical activity (see protocol of physical activity).
Self-reported information during follow-up. At the follow-up visits, participants are asked
about clinical events that may have occurred between visits and information about
medication prescribed is updated.
Evaluation of adverse effects: At 6 months and yearly thereafter, participants complete
a specific questionnaire to report any adverse effects felt to be derived from the
intervention or weight loss.
Electrocardiogram: ECGs are performed at the primary care centers at the first screening
visit, 6 months, and at annual follow-up visits thereafter. The ECGs are scanned, stored
and registered in the specific database designed for that purpose. The nursing staff at
each recruiting center will be responsible for receiving and scanning the ECGs, digitizing
their contents, and maintaining the registry and database.
Fasting blood collection: Fasting blood samples are collected at the baseline visits, 6
months, 12 months, 3 years, 5 years, 7 years, and at the final follow-up visit.
Conventional analyses [lipid profile (total cholesterol, HDL-cholesterol, LDL-cholesterol,
and triglycerides), fasting plasma glucose, blood cell count, serum sodium, potassium,
calcium, uric acid, urea, creatinine, albumin, C-reactive protein, erythrocyte
sedimentation rate, hemoglobin A1C, liver function tests (serum bilirubin, alkaline
phosphatase, alanine transaminase, aspartate aminotransferase and gamma-
glutamyltranspeptidase) and optional coagulation tests (prothrombin time, activated
partial thromboplastin time and fibrinogen)] are performed at the baseline and follow-up
visits according to the trial protocol. The nursing staff at the recruiting centers is
responsible for collecting, processing, delivering, storing and preserving the samples,
digitizing the information, and maintaining the registry and database for all samples and
analyses (see protocol of biological sample collection).
Morning spot urine collection: A sample of morning spot urine is collected in situ at the
baseline visit, 6 months, 12 months, 3 years, 5 years and 7 years, and at the final follow-
up visit. Conventional analyses (albumin and creatinine in urine) are performed at the
baseline and follow-up visits according to the trial protocol. The nursing staff is
responsible for collecting, processing, delivering, preserving, recording, and maintaining
the samples.

23
Nail sample collection: Nail samples are collected in situ at the baseline visit, 12 months,
3 years, 5 years and 7 years and at the final follow-up visit. Patients are asked to attend
the visits without having cut their toenails. Using toenail clippers, the patients’ nails are
cutted and placed in labelled zip-lock bags.
Neuropsychological and Quality of Life evaluation:
This evaluation includes three parts:
A) Cognitive Function
B) Quality of life
C) Psychopathology
At baseline and every two years thereafter (2-, 4-, 6- and 8-year follow-up visits),
participants complete a battery of 6 tests of Cognitive Function (see below, section A).
The first (Mini-Mental State Examination (MMSE) is a general screening cognitive test,
while the other 5 tests explore different cognitive domains and are aimed at assessing
changes in cognitive performance. The Cognitive Function tests will be alternated with
the Quality of life tests (see section B), so that in even years the 6 tests of Cognitive
Function will be collected and in odd years only the quality of life test will be administered
(see below, section B). The 2-year lapse between sequential cognitive tests will reduce
bias due to a "learning" effect.
The Quality of Life scales (Short -Form 36 or SF -36, see section B) are collected from
all participants at the beginning of the study and in odd years thereafter (after 1-, 3-, 5-,
and 7- year follow-up visits), while the psychopathology questionnaires (see below,
section C) are collected at the beginning of the study and annually.

All instruments included in the Cognitive battery (A), Quality of Life (B), and
psychopathology (C) have been standardized for the Spanish population in the age
range of the study. The complete battery of cognitive, quality of life, and psychopathology
examinations includes the following tests:

A) Six cognitive neuropsychological tests (lasting 16 minutes, to be completed in face-

to-face interviews):
1) MMSE (Folstein et al, 1975);
2) Semantic verbal fluency test: “animals in 1 minute” (Ramier and Hécaen, 1970, 1977;
Benton et al., 1994);
3) Phonemic verbal fluency test: “words in 1 minute starting with the letter ‘p’ (Benton et
al., 1994);
4) Verbal and visual working memory: reverse digits test (WAIS-III), Wechsler, 1997):

24
5) Trail Making Test (Reitan, 1973);
6) Clock test (Clock drawing test or CDT).

Normative data for these tests in the Spanish population have been published by Peña-
Casanova et al. (2009a, 2009b). These six tests are collected in the run-in period and
each even follow-up year thereafter (years 2, 4, 6 and 8). These six tests, which take
roughly 16 minutes to complete, are administered by PREDIMED-PLUS personnel at the
third screening visit and each even follow-up year thereafter (years 2, 4, 6 and 8).

B) One test of Quality of Life (5-10 minutes, to be completed at home):

The SF-36 (36-item) quality of life questionnaire (Alonso et al., 1995, 1998; Ware and
Gandek 1998) is administered during the run-in period and every odd year of follow-up
thereafter (years 1, 3, 5 and 7). In this way, these tests will be alternated with the
neuropsychological questionnaires.

C) Three Psychopathological questionnaires (lasting 20-25 minutes, to be completed at

home):
1) Beck Depression Inventory (BDI-II) (Beck, Steer and Brown, 1996; Sanz, Navarro
and Vázquez, 2003);
2) Multidimensional scale of weight locus control (Wallston, Wallston and DeVellis,
1978);
3) Screening for comorbid eating disorders with diagnostic criteria (DSM-IV-TR; APA,
2000).

The 10 questionnaires above (sections A, B and C) are collected in all participants in the
PREDIMED-PLUS study.
The four questionnaires of sections B) and C) are delivered to the participants at the
screening visit 1 to be completed at home or at another time outside the study visit.
Participants are required to deliver filled-in questionnaires to the recruiting centers within
a 15-day period. The same procedure is repeated at follow-up visits when required. The
nursing staff at each recruiting center is responsible for collecting, processing, sending,
and keeping all the information pertaining to the cognitive tests.
To ensure that graphical data from cognitive tests (drawings of the MMSE and clock and
Trail Making tests) are saved for future monitoring. The questionnaires from Group A, B
and C tests are collected in paper format, taking advantage of the optical scanning forms
for the MMSE, the clock test and the FS-36 designed for this purpose. Once completed,

25
the MMSE, the clock test and the SF-36 are mailed to the Navarra center (after saving a
security photocopy in the recruiting center) to be computerized by optical reading, a time-
saving and materially mistake-free procedure.

9. Outcome definition and ascertainment

Clinical events are ascertained by a Clinical Event Ascertainment Committee led by
Dr. Fernando Arós of the Vitoria group. The committee members are M. Aldamiz, A.
Alonso, J. Berjón, L. Forga, J. Gállego, M. A. García Layana, A. Larrauri, J. Portu, J.
Timiraos, and M. Serrano-Martínez. Clinical event ascertainment are based on
information collected from the participants’ medical records, which each year are
reviewed on an ad hoc basis by the medical doctors participating in the PREDIMED-
PLUS trial. These doctors and the members of the Ascertainment Committee will be
blinded to the assignment of participants to the two intervention groups. The reports sent
to the Clinical Events Committee contain no personal information about the participants
and are identified only by a code.

9.1. Primary outcomes

1. Non-fatal acute coronary syndrome (acute myocardial infarction), non-fatal stroke or
cardiovascular mortality.
1.a. Acute myocardial infarction (MI) are defined according to the third universal definition
of MI on behalf of the Joint ESC/ACCF/AHA/WHF Task Force (Thygesen et al., 2012)
as evidence of myocardial necrosis in a clinical setting consistent with acute
myocardial ischemia.

Any one of the following criteria meets the diagnosis for MI:
• Detection of a rise and/or fall of cardiac biomarker values [preferably cardiac
troponin (cTn)] with at least one value above the 99th percentile upper reference
limit (URL)
AND
• At least one of the following:
(i) Symptoms of ischemia.
(ii) New or presumed new significant ST-segment–T wave (ST–T)
changes or new left bundle branch block.
(iii) Development of pathological Q waves in the ECG.
(iv) Imaging evidence of new loss of viable myocardium or new
regional wall motion abnormality.

26
 (v) Identification of an intracoronary thrombus by angiography.
Prior MI
Any one of the following criteria meets the diagnosis for prior MI:
• Pathological Q waves with or without symptoms in the absence of non-ischemic
causes.
• Imaging evidence of a region of loss of viable myocardium that is thinned and fails
to contract, in the absence of a non-ischemic cause.
• Pathological findings of a prior MI

1.b. Stroke is defined as an acute neurological deficit lasting more than 24 hours caused
by an abrupt impairment of brain function due to blockage of blood flow in a particular
artery supplying the brain (thrombosis or arterial embolism) or a cerebral
haemorrhage.

Ischemic Stroke is defined following the updated definition of stroke for the 21st
Century: A Statement for Healthcare Professionals from the American Heart
Association/American Stroke Association (Sacco RL, et al. 2013) as an episode of
neurological dysfunction caused by focal cerebral, spinal, or retinal infarction.
Central nervous system (CNS) infarction is brain, spinal cord, or retinal cell death
attributable to ischemia, based on:
1. Pathological, imaging, or other objective evidence of cerebral, spinal cord, or
retinal focal ischemic injury in a defined vascular distribution;
2. Clinical evidence of cerebral, spinal cord, or retinal focal ischemic injury
based on symptoms persisting ≥24 hours or until death, and exclusion of
other potential causes such as hypoglycaemia or seizures.
Silent CNS infarction are not considered as a primary end-point if defined as
imaging or neuropathological evidence of CNS infarction without a history of acute
neurological dysfunction attributable to the lesion.

Haemorrhagic Stroke. Stroke caused by intracerebral hemorrhage is defined as

rapidly developing clinical signs of neurological dysfunction attributable to an
intracerebral hemorrhage, defined as a focal collection of blood within the brain
parenchyma or ventricular system that is not caused by trauma. Stroke caused by
subarachnoid hemorrhage is defined as a rapidly developing signs of neurological
dysfunction and/or headache because of bleeding into the subarachnoid space,
which is not caused by trauma.

27
 Silent cerebral hemorrhage is not considered as primary end-point. It is defined as
a focal collection of chronic blood products within the brain parenchyma,
subarachnoid space, or ventricular system detected at neuroimaging or
neuropathological examination that is not caused by trauma and without a history of
acute neurological dysfunction attributable to the lesion.

1.c. Cardiovascular mortality: Includes sudden death and non-sudden cardiovascular

death (Buxton AE, et al. 2006).

Sudden (cardiac) death is due to cessation of cardiac activity with hemodynamic

collapse, typically due to sustained ventricular tachycardia/ventricular fibrillation. It
may be:
— Witnessed instantaneously in a previously stable patient. This may occur with or
without preceding signs or symptoms, or may occur immediately following sudden
dyspnea, light-headedness, or palpitations.
— Unwitnessed. Patient found dead who at the time of last witnessed contact was
in his/her usual state of health without medical complaints or obvious difficulty.
This applies to patients dying during sleep.

Non-sudden cardiac death: Includes deaths of patients from acute pulmonary

edema with severe, progressive heart failure, cardiogenic shock, or after a recent
cardiac surgical procedure.

Non-cardiac vascular death: Includes deaths due to thromboembolic events, stroke,

dissecting aneurysm and peripheral artery disease.

2. Weight change. The study nurse records weight at each follow-up visit. The
measurement are made according to the study manual of operations and with
participants dressed in light clothing and no shoes and accessories.

9.2. Secondary outcomes

1. Total mortality. This endpoint comprises all causes of death, including those from
CVD (see point 1c of primary end-point), as well as trauma, renal failure,
neoplasia, sepsis, suicide and death of undetermined cause. All deaths should be
confirmed by reviewing the National Death Index.

28
2. Changes in waist circumference. The study nurse measures waist circumference at
each follow-up visit according to the manual of operations.

3. Non-ST-segment elevation acute coronary syndrome (unstable angina): The

diagnosis of unstable angina is made following the definition of the ESC Guidelines
for the management of acute coronary syndromes in patients presenting without
persistent ST-segment elevation (Hamm et al, 2011); It requires the presence of at
least one of the following clinical characteristics:
a. Prolonged (>20 min) anginal pain at rest.
b. New onset (de novo) angina (Class II or III of the Classification of the Canadian
Cardiovascular Society).
c. Recent destabilization of previously stable angina with at least Canadian
Cardiovascular Society Class III angina characteristics (crescendo angina).

4. Coronary revascularization (percutaneous or surgical): The two main indications for

percutaneous or surgical revascularization are:
1) Patients with unstable angina or non-ST-segment elevation acute coronary
syndrome.
2) Patients considered likely to benefit from such surgery on the basis of the location
and severity of chest pain, the number of vessels affected, and the presence of
left ventricular dysfunction (Hamm et al, 2011).

5. Heart failure. Acute and chronic heart failure (HF) is a syndrome in which patients
have typical symptoms and signs resulting from an abnormality of cardiac structure
or function (McMurray JJ, et al. 2012; Yancy CW, et al. 2013). The cardinal
manifestations of HF are dyspnea and fatigue, which may limit exercise tolerance,
and fluid retention, which may lead to pulmonary and/or splanchnic congestion and/or
peripheral edema.
5.a. The diagnosis of HF with Reduced Ejection Fraction requires three conditions to
be satisfied: 1. Symptoms typical of HF; 2. Signs typical of HF and 3. Reduced
ejection fraction (< 40%)
5.b. The diagnosis of HF with Preserved Ejection Fraction requires four conditions to
be satisfied: 1. Symptoms typical of HF; 2. Signs typical of HF; 3. Normal or only
mildly reduced left ventricular ejection fraction and non-dilated left ventricle; and
4. Relevant structural heart disease (left ventricular hypertrophy/left atrium

29
 enlargement) and/or diastolic dysfunction
5.c. A Heart Failure event may include hospitalization or an urgent outpatient visit. In
this setting the event needs to meet ALL of the following criteria:
- The patient exhibits documented new or worsening symptoms of HF on
presentation, including at least ONE of the following: Dyspnea, decreased
exercise tolerance, fatigue or other symptoms of worsened end-organ perfusion
or volume overload.
- The patient has objective evidence of new or worsening HF, consisting of at least
TWO physical examination findings OR one physical examination finding and at
least ONE laboratory criterion), including: Physical examination findings
considered to be due to heart failure, including new or worsened peripheral
edema, increasing abdominal distention or ascites (in the absence of primary
hepatic disease), rales/crackles/crepitations at pulmonary auscultation,
increased jugular venous pressure and/or hepatojugular reflux, S3 gallop, and
clinically significant or rapid weight gain thought to be related to fluid retention
- Laboratory evidence of new or worsening HF, if obtained within 24 hours of
presentation, including: Increased B-type natriuretic peptide (BNP)/ N-terminal
pro-BNP (NT-proBNP) concentrations OR cardiological evidence of pulmonary
congestion OR echocardiographic data of congestion or decreased cardiac
output.
- The patient receives initiation or intensification of specific treatment for HF.

6. Peripheral artery disease. Ascertainment is made according to the Inter-Society

Consensus for the Management of Peripheral Arterial Disease (TASC II) (Norgren
et al., 2007) and ESC Guidelines for the diagnosis of peripheral artery disease
(Tendera M, et al., 2011). For participants with intermittent claudication, aged 60-69
with one cardiovascular risk factor, or aged ≥70 years and a resting ankle-brachial
systolic pressure index ≤0.90, or an abnormal echo-Doppler examination, magnetic
resonance imaging, or arteriography are considered as diagnostic (confirmed case).
7. Venous thromboembolism (VTE): all VTE need to satisfy the standard diagnosis
criteria for venous thrombosis or (thromb-) Pulmonary Embolism (PE) in the general
population (see below 1-3). The diagnosis should be confirmed by objective imaging
techniques (including echography, phlebography, pulmonary computed tomography
angiography (angioCTA), NMR, etc.) and not only be based on the clinical suspicion.
Standard diagnosis criteria for VTE in clinical studies (Carrier M et al, 2012):
1. Deep venous thrombosis, defined as the loss of venous compressibility or the inability

30
 of filling the deep vein intraluminal segment at the lower/upper limbs, as detected by
echography with venous compression or phlebography, respectively.
• The presence of thrombus at the distal lower limb (distal from the popliteal vein)
qualifies for primary VTE only if it is asymptomatic.
• All proximal thrombus qualify for final primary end-point if detected by imaging
techniques (echography or radiology), regardless of whether it is or not asymptomatic.
2. Pulmonary Embolism (PE) is defined as:
Contrast pulmonary arteriography:
• Defects in intraluminal filling, as contrasted with two projections.
• Sudden stoppage of the contrast in one or several vessels with a diameter greater
than 2.5 mm
• Pulmonary scintigraphy based on ventilation/perfusion (V/Q):
o A V/Q-pulmonary scintigraphy with high probability of PE in patients with no low
clinical probability of PE.
• Pulmonary angiography using computed tomography:
o Defects in filling sub-segmental or more proximal vessels
3. Fatal PE is defined as:
• Death exclusively caused by PE and/or its confirmation at autopsy or using radiology
techniques
Important considerations:
a) Superficial venous thrombophlebitis should not be described as VTE.
b) It is highly recommended to describe VTE according to the anatomic position:
• Lower limbs
• Upper limbs
• Pulmonary embolism
• Others: vessels at the splanchnic level, cerebral veins, etc.
c) The description of the VTE is highly convenient (for instance, distal to popliteal vein
vs. proximal VTE; sub-segmental level vs. central PE)
d) VTE associated with a central catheter (for instance, deep venous thrombosis at the
upper limbs) should be reported separately.
e) Incidental VTE should be differenced from any other symptomatic events.

8. Atrial fibrillation (AF): AF is defined following the Guidelines of the American College
of Cardiology Foundation/American Heart Association Task Force on Practice
Guidelines together with the European Society of Cardiology, the European Heart
Rhythm Association, and the Heart Rhythm Society (Camm AJ, et al. 2010; Fuster V,

31
 et al. 2011), as a cardiac arrhythmia with the following characteristics:
(1) The surface ECG shows ‘absolutely’ irregular RR intervals, i.e., RR intervals that
do not follow a repetitive pattern.
(2) There are no distinct P waves on the surface ECG. Some apparently regular atrial
electrical activity may be seen in some EKG leads, most often in lead V1.
(3) The atrial cycle length (when visible), i.e., the interval between two atrial
activations, is usually variable and <200 ms (>300 bpm).

9. Type 2-diabetes. New-Onset Type 2 Diabetes cases are diagnosed following the
recommendations of the American Diabetes Association:
1. HbA1C ≥6.5%. This test should be performed in a laboratory using a method that
is National Glycohemoglobin Standardization Program (NGSP) certified and
standardized to the DCCT assay. OR
2. Fasting plasma glucose (FPG) ≥126 mg/dL (7.0 mmol/L). Fasting is defined as no
caloric intake for at least 8 hours OR
3. Two-hour plasma glucose (PG) ≥ 200 mg/dL (11.1 mmol/L) during an oral glucose
tolerance test (OGTT). This test should be performed as described by the WHO,
using a glucose load containing the equivalent of 75 g anhydrous glucose
dissolved in water OR
4. In a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a
random plasma glucose ≥200 mg/dL (11.1 mmol/L).
In the absence of unequivocal hyperglycemia, results should be confirmed by repeat
testing (American Diabetes Association, 2014).

10. Type-2 diabetes complications. Participants are assessed yearly for microvascular
complications of diabetes:
1. Diabetic nephropathy: Kidney disease in diabetes is defined based on the
alteration of glomerular filtration rate (GFR) and /or the presence of persistent
albuminuria at levels of 30 mg/24 h or more (normal albumin excretion is currently
defined as < 30 mg/24 h). GFR is estimated through a quantitative formula, the
Modification of Diet in Renal Disease (MDRD) equation, that measures the
progression of kidney involvement. Persistent albuminuria is determined by the
urine albumin to creatinine ratio (normal <30 mg albumin/g creatinine) in a routine
morning urine sample. Because of variability in urinary albumin excretion, two of
three morning specimens collected within a 3- to 6-month period should be
abnormal before considering a patient to have developed increased urinary

32
 albumin excretion or a progression of albuminuria. The presence of one or two of
the above criteria indicates renal disease in these patients and the requirement
for appropriate follow-up for progression of renal disease (American Diabetes
Association, 2014).
The stages of chronic kidney disease by GFR are reported as follows:
1. Kidney damage with normal or increased GFR ≥90 mL/min/1.73 m2 body
surface area
2. Kidney damage* with mildly decreased GFR 60–89 mL/min/1.73 m2 body
surface area
3. Moderately decreased GFR 30–59 mL/min/1.73 m2 body surface area
4. Severely decreased GFR 15–29 mL/min/1.73 m2 body surface area
5. Kidney failure <15 mL/min/1.73 m2 body surface area or dialysis
2. Diabetic retinopathy: Diagnosed by ophthalmologic examination and/or treatment
with laser photocoagulation (American Diabetes Association, 2014).
3. Diabetic polyneuropathy: Diagnosed by clinical symptoms, neurological
examination and results of electrophysiological studies of peripheral nerves
(American Diabetes Association, 2014).
*Kidney damage defined as abnormalities in urine, blood, or imaging tests.

11. Cancer. All cancers except non-melanoma skin cancer are considered. Cancer cases
are coded according to the International Classification of the World Health
Organization (International Agency for Research in Cancer, WHO, 2014).

12. Dementia/Alzheimer’s disease. Cases are ascertained according to the

Recommendations from the National Institute on Aging and the Alzheimer´s
Association workgroup (McKhann et al., 2011) or if a diagnosis of dementia is
reported by a neurologist.

13. Other dementias: Cases are ascertained according to McKhann et al. 2011 criteria
(see below) or if a diagnosis of dementia is reported by a neurologist.
Dementia is diagnosed when there are cognitive or behavioral (neuropsychiatric)
symptoms that: 1. Interfere with the ability to function at work or at usual activities;
and 2. Represent a decline from previous levels of cognitive functioning; and 3. Are
not explained by delirium or major psychiatric disorder; 4. Cognitive impairment is
detected and diagnosed through a combination of (1) history-taking from the patient

33
 and a knowledgeable informant and (2) an objective cognitive assessment; 5. The
cognitive or behavioral impairment involves a minimum of two domains).

14. Parkinson’s disease. Cases are ascertained according to the diagnostic criteria
described by Hughes et al (Hughes AJ, et al., 1992) or if reported by a neurologist.

15. Unipolar depression. The diagnosis must be made according to the DSM-V criteria
(American Psychiatric Association, 2013). In this, definition major depression,
persistent depression, and other depressions included in Depressive Disorders
(DSM V) are accepted. Diagnosis of depression made by primary care physicians
or psychiatrist in participants treated with antidepressant drugs for more than 6
moths is accepted. If this is the case, ICD 10 (International Statistical Classification of
Diseases and Related Health Problems, 10th version) diagnosis of depressive
episodes are also accepted. For physicians and psychiatrists not using ICD 10 or
DSM V, a positive response to the two questions included in the NICE clinical
guidelines is recommended (https://www.nice.org.uk).

16. Osteoporotic fractures. Low-energy fracture is defined as the fracture produced by

a same-level fall. Fractures are identified from X-rays reports obtained from at least
two radiological reports. High trauma fractures, potentially pathological fractures
(e.g., cancer or Paget’s disease), or fractures of the head, fingers and toes are not
considered (Bliuc D, et al. 2009).

17. Gallstone disease or cholecystectomy: Gallstone disease isdiagnosed according to

the findings obtained by imaging techniques including abdominal ultrasonography,
computed tomography or magnetic resonance imaging. Diagnosis of
cholecystectomy require the corresponding surgical report.

18. Symptomatic gout: Defined following the criteria of the American College of
Rheumatology. Typically, the disease first presents as arthritis that is acute and
episodic, but can be recurrent. Gout can also present as chronic arthritis of one or
more joints. This clinical picture is built on a foundation of an excess body burden of
uric acid, manifested in part by hyperuricemia, which is defined as serum uric acid
levels greater than 7.0 mg/dL (Khanna D, et al. 2012).

34
19. Transient Ischemic Attack: The diagnosis must be made according to the Scientific
Statement of the American Heart Association/American Stroke Association Stroke
Council (Easton JD et al. 2009): a transient episode of neurological dysfunction
caused by focal brain, spinal cord, or retinal ischemia, without acute infarction
demonstrated by neuroimaging, preferably magnetic resonance imaging
techniques.

20. Cataracts surgery: Defined by a medical report of cataracts surgery.

21. Surgery for obesity: Defined by a medical report of bariatric surgery.

9.3. Intermediate markers

Changes in nutrient intake and dietary patterns will be determined by changes in the
17-item score of adherence to the energy-restricted Mediterranean diet (intensive
intervention group) or the 14-item score (control group) and by changes in food and
nutrient intake determined by the 143-item food frequency questionnaire administered
during follow-up.
Changes in systolic and diastolic blood pressure, serum lipid concentrations, fasting
glucose levels, renal function, uric acid, hemoglobin A1C, C-reactive protein, and liver
function are evaluated yearly for the duration of the intervention.
Yearly, are also evaluated the percentage of participants in each group requiring
anti-hypertensive, anti-diabetic or lipid-lowering medication, results of ECGs, cognitive
function, quality of life, and psychological and neuropsychological questionnaire scores.

10. Randomization procedure (random assignment)

Between one and four weeks after the third screening visit, each recruiting center
randomized assign eligible candidates to one of two groups, intensive intervention group
or usual care (control) group, using a centrally-controlled, computer-generated random-
number system (available at: www.predimedplus.com). The coordinating center was
responsible for the randomization procedure by which participants wererandomized
assigned with stratification by center, sex, and age group (<65, 65-70, >70 years).
Married or unmarried couples were randomized together. The recruiting centers enter
the participants’ identification criteria into the internet-based system. The system then
automatically assigns the participants or partners of participants to their groups. Once
this occurs, the group assigned cannot be changed. In the specific cases of couples in

35
which the spouse was recruited at different times, the last spouse entering the study was
assigned (not randomised) to the same study arm than his/her partner in order to ensure
high adherence to the intervention and avoid contamination and potential conflicts
between partners of the same household.

11. Intervention protocol

All participants will continue to receive usual healthcare from their family doctors and
primary care physicians throughout the duration of the trial. At no time will PREDIMED-
PLUS personnel deliver medical care.

11.1. Phases of the study for participants assigned to the intensive intervention program
with energy-restricted Mediterranean diet (intervention group)

First six months

In addition to the initial visit, participants assigned to the intensive intervention
program took part in six individual sessions (I) and 6 group sessions (G) in the first six
months (see below):

Month 1 Month 2 Month 3 Month 4 Month 5 Month 6

G I G I G I G I G I G I

Participants also received a third monthly contact by way of a telephone call

from a dietitian aimed at reinforcing the trial’s objectives and answering any queries.
During these first six months, participants in the intervention group were
encouraged to aim for a reduction in their initial weight of 10% and a reduction in their
initial waist circumference of between 5 and 10%. The aim of the trial during these first
six months was that the average weight loss of the participants in the intervention group
was above 8% and the average waist circumference reduction was above 5%. Success
in achieving an initial weight loss is known to be a predictor of long-term weight loss. For
this reason, all participants were given a chart on which to record and correctly monitor
their own weight and waist circumference.
During this period they were encouraged to substitute one meal for low-calorie
foods and so were offered a wide range of pleasant alternatives in keeping with the
culinary traditions of the Mediterranean diet (see below).
Finally, if by the final visit the participant had still been unable to reach the
objectives established for this phase of the trial (month 6), he or she took part in a

36
motivational interview session with the dietitian in order to determine why he/she had
not reached his or her weight-loss goal (see below), try to readdress the situation, and
provide appropriate rescue measures.

Months 7-12
Participants attended one individual session (I) and one group session (G) every
month in months 7 to 12 of the trial.

YEAR 1 Month 7 Month 8 Month 9 Month 10 Month 11 Month 12

Months 7-12 G I G I G I G I G I G I

They also received a third contact every month by way of a telephone call from
a dietitian aimed at reinforcing the trial’s objectives and answering any queries.
The first-year follow-up visit (see below) coincided with the last individual visit for
this phase.

Years 2-6
After the first year and in each of the remaining years of the trial (years 2-6), the
participants are attending one quarterly individual session (I) and one monthly group
session (G) and are receiving two quarterly telephone calls (T), in accordance with the
table below:

Mont Month Month Mont Month Month Mont Month Month Mont Month Month Mont
h 13 14 h 15 16 17 h 18 19 20 h 21 22 23 h 24

13- G T G T G I G T G T G I G T G T G I G T G T G I
72

Months 25-36 follow the same procedure as months 13-24, and this procedure will
be repeated for the successive years.
The annual follow-up visits (see below) coincide with the last individual session of
each year (month 24 above). Throughout the trial, any missed visits are reprogrammed.

11.2. Program of individual and group sessions for participants assigned to the energy-
restricted Mediterranean diet
A) Individual visits

37
All individual visits comprise:
i) Distribution of a 17-item questionnaire of adherence to an energy-restricted
Mediterranean diet.
ii) Weight and waist circumference measurement by a dietitian.
iii) An individual motivational interview with the dietitian in accordance with the changes
in weight observed and the participant’s scores on the 17-item adherence to
Mediterranean diet questionnaire (see below).
iv) Encouragement to self-monitor weight and waist circumference. Participants are
provided charts for self-registering and self-monitoring weight and waist
circumference in accordance with the Body Weight Simulator of the National Institute
of Diabetes and Digestive and Kidney Diseases (Hall et al., 2011; National Institute
of Diabetes and Digestive and Kidney Diseases, 2012). This simulator is also
provided to participants at the first individual session with use instructions.
v) Personalized recommendations for increasing physical activity.
B) Group sessions
At these group sessions participants are provided shopping lists, menus, recipes,
descriptions of typical components of the Mediterranean diet and advice on lifestyle
changes. PREDIMED-PLUS dietitians lead these sessions, which are attended by no
more than 20 participants. The sessions comprise:

i) An introductory talk to review the 17-item questionnaire on adherence to the energy-

restricted Mediterranean diet (see below).
ii) A 15-minute presentation of the main aspects of the Mediterranean diet with
audiovisual material prepared by the coordinating center.
iii) Answers to any queries on any aspect of the intervention.
iv) Delivery of the following documents:
• Description of 4-5 low-calorie foods typical of a Mediterranean diet and adapted to
the season.
• Weekly food shopping list adapted to the season.
• Weekly meal plan (with detailed menus) adapted to the shopping list.
• Recipes for the suggested menus.
v) Delivery of gratis virgin olive oil (one liter per month) and nuts (125 g per month) to
each participant.
vi) At the end of the session participants are reminded of the date of the next session.
11.3. Program of individual and group sessions for participants assigned to the control
group.

38
Control group participants receive usual medical care from medical staff at their health
institutions. The importance of their attending usual medical visits is stressed to them.
Participants receive all the written information related to the Mediterranean diet used in
the PREDIMIED trial as well as leaflets with general lifestyle recommendations for
managing the metabolic syndrome. At the beginning of the study, a group session and
an individual session is held at which dietitians deliver documents similar to those used
in the PREDIMED trial (shopping lists, recipes, menus, and descriptions of
Mediterranean diet components). The dietitians do not provide participants in the control
group with instructions on how to lose weight, as this is the responsibility of their family
doctors or specialists (usual care). They are also offered a group session every 6
months. At the initial visit and at each 6-month group session, participants are provided
free virgin olive oil (6 liters every 6 months) and nuts (750 g every 6 months). In order to
encourage compliance with the trial, supply of olive oil and nuts to the participants is
contingent on their attending these sessions. The 6-month group sessions include tips
on how to follow the Mediterranean diet to prevent CVD but advice on calorie restriction,
weight loss or increased physical activity is not given and no such objectives are
entertained.

11.4. Dietary and lifestyle intervention

The Intervention Committee led by Jordi Salas-Salvadó coordinates the dietary and
lifestyle intervention. This committee is made up of four coordinators (Jordi Salas-
Salvadó, Montse Fitó, Ramón Estruch and Miguel Ángel Martínez-Gonzalez), three of
whom are responsible for the three intervention sub-committees: Dietary Intervention
(chair: Jordi Salas-Salvadó; members: Nancy Babio, Emilio Ros and Ana Sánchez-
Tainta); Physical Activity (chair: Montse Fitó; members: Helmut Schröder, Ascensión
Marcos, Miguel A. Martínez-González, Dolores Corella, and Julia Warnberg); and
Behavior Treatment (chair: Ramon Estruch; members: Fernando Fernández-Aranda,
Cristina Botella and Jordi Salas-Salvadó). This Committee is responsible for designing
the lifestyle intervention program for the intensive intervention group and ensuring that it
is implemented correctly. Miguel Ruiz-Canela, Miguel A. Martínez-González and Jordi
Salas-Salvadó are responsible for ethical considerations.

Dietary recommendations
Many aspects of a diet’s quality can affect body weight and the risk of obesity-related
illnesses to a greater extent than relative macronutrient content (Mozaffarian et al., 2011;

39
Ludwig, 2012). In recommendations given to participants, two food groups (A and B) are
clearly differentiated:
A) Traditional dietary patterns based on whole foods or minimally processed foods, such
as the Mediterranean diet, which incorporates many cardioprotective foods and few
harmful ones. The consumption of virgin olive oil, nuts (especially walnuts), fruits and
vegetables, salads, whole grains, fiber-rich foods and low-fat yogurts have been
consistently associated with weight loss or lower weight gain (Martinez-Gonzalez,
Bes-Rastrollo, 2011; Mozaffarian et al., 2011).
B) On the other hand, sugar-sweetened beverages, fast foods, refined grain products
(especially white bread, which is widely consumed in Spain), white rice, pasta (except
for whole-grain pasta), French fries, potatoes, trans fats (mainly present in
commercial bakery products in Spain), sweets, cakes, pies, sugar, precooked meals,
sausages or cold cuts of processed meats, and patés have been consistently
associated with weight gain (Schulze et al., 2006; Mozaffarian et al., 2011).

The main focus of the intensive intervention program (intervention group) lies
therefore in the diet’s overall quality, with the aim of avoiding foods from the B group and
replacing them with foods from the A group.
In addition, by taking into account energy requirements estimates according to the
Institute of Medicine equation as well as the participants’ basal metabolic rate and level
of physical activity, a reduction in energy intake of roughly 600 kcal (about 30% of
estimated energy requirements) is envisaged. The energy-restricted Mediterranean diet
involves reduced consumption of meat and cold cuts, sugars, white bread, processed
fruit juices and sugary beverages, and other foods from the B group, as follows:
ENERGY-RESTRICTED MEDITERRANEAN DIET
NUTRIENT RECOMMENDED INTAKE
Calories1 Reduction of ≈600 kcal/day (about 30%) from usual intake
Total fat2 35-40 % of total calories
Saturated Fatty Acids 8-10 % of total calories
Monounsaturated Fatty Acids > 20 % of total calories
Polyunsaturated Fatty Acids > 10 % of total calories
Cholesterol3 < 300 mg/day
Proteins4 Approximately 20 % of total calories
Carbohydrates5 40-45 % or more of total calories (of low glycemic index)
No more than 100 mmol/day (roughly 2.4 g of sodium or
Sodium chloride
roughly 6 g of sodium chloride)
Dietary fiber 30-35 g/day

1. A reduction in calories of 500 to 1,000 kcal/ day help to achieve a weight loss of 0.5
to 1 kg/week.

40
Alcohol provides unnecessary calories and displaces the intake of more nutrient-dense
foods. Not only does the consumption of alcohol increase the number of calories in one’s
diet but in epidemiological and experimental studies it has also been associated with
obesity. For this reason, although the 17-item adherence to the Mediterranean diet
questionnaire contains one item for the consumption of wine, the impact of calories from
alcohol on the overall calorie intake ise carefully evaluated and monitored and the
consumption of alcoholic beverages other than wine should be avoided.

2. The consumption of wine permitted is one or two glasses per day for women and two
or three glasses per day for men. The consumption of other sources of alcohol other than
wine is discouraged. Red wine is preferred over other types and it is recommended that
the wine be consumed at mealtimes (Gea et al., 2014).

3. Fat restriction involves fat from animal foods. Olive oil and nuts must be the preferred
sources of fat.

4. Proteins must be derived first from plant and second from lean animal sources (like
fish or poultry).

5. Carbohydrates must be derived from solid, minimally processed and fiber-rich foods
with a low glycemic index, such as vegetables, fruits and whole grains, all of which are
good sources of vitamins, minerals, and fiber. A diet that is rich in soluble fiber such as
oat bran, legumes, and most fruits and vegetables may be effective in reducing blood
cholesterol levels and insulin resistance. A diet that is high in all types of fiber may also
help to control weight by promoting satiety and maintaining lower levels of total energy
intake.

6. During weight loss, attention must be given to maintaining an adequate intake of

vitamins and minerals. Maintaining the recommended calcium intake of 1,000 to 1,500
mg/day is especially important for postmenopausal women who may be at risk of
osteoporosis.

41
 Participants in the intensive intervention group receive counseling to help them
progressively increase their compliance with the following 17 objectives (the 17-item
questionnaire on adherence to the energy-restricted Mediterranean diet). One
point is awarded for each objective met:

1. Use only extra-virgin olive oil for cooking, salad dressings, and spreads.
2. Consume ≥3 portions of fruit per day.
3. Consume ≥ 2 portions of vegetables/garden produce per day (at least 1 portion raw
or in a salad).
4. Reduce consumption of white bread to d1 serving/day (1 serving = 75 g).
5. Consume whole grain cereals and pasta ≥5 times per week.
6. Consume d1 serving (1 serving = 100-150 g) of red meat, hamburgers, or meat
products (ham, sausage, etc.) per week.
7. Consume less than 1 serving of butter or cream per week (1 serving = 12 g).
8. Consume less than one sugary beverage or sugar-sweetened fruit juice per week.
9. Consume ≥3 servings of legumes per week (1 serving = 150 g).
10. Consume ≥3 servings of fish or shellfish per week (1 serving = 100-150 g fish, or 4-
5 units or 200 g shellfish).
11. Consume < 3 sweets or pastries, such as cakes, cookies, sponge cake, or custard,
per week.
12. Consume ≥ 3 servings of nuts (including peanuts) per week (1 serving = 30 g).
13. Consume chicken, turkey or rabbit meat instead of beef, pork, hamburgers or
sausages.
14. Use sofrito (sauce made with tomato and onion, leek or garlic, simmered in olive oil)
≥ 2 times per week.
15. Do not add sugar to beverages (coffee, tea); instead, replace sugar with non-caloric
artificial sweeteners.
16. Reduce consumption of pasta or rice <3 servings per week (unless the pasta or rice
are whole grain products).
17. Consume 2-3 glasses of wine (200 mL) per day (men) or 1-2 glasses of wine per day
(women). This item will be only promoted among participants who were consumers of
alcohol at baseline. Abstainers will never be invited to start consuming wine or any other
alcoholic beverage.

The intervention tool for the control group, on the other hand, is the PREDIMED 14-
item adherence questionnaire to the non-energy-restricted Mediterranean diet

42
(Schroeder et al., 2011). However, the 17-item questionnaire is also collected in control
group participants for comparison purposes.

Physical exercise recommendations

Participants are encouraged to gradually increase their level of physical activity to at
least 45 minutes per day (6 days per week) after 6 months of intervention and their
progress is monitored. The physical activity program includes aerobic activities, such as
gentle walking or any equivalent activity of moderate intensity and resistance training
(Fernández et al., 2012). The dietitians adapt their recommendations to personal
preferences and encourage participants to switch between activities with the same
metabolic equivalence of tasks.

Psycho-behavioral therapy
Participants are instructed on strategies and provided tools for solving problems
associated with consuming high calorie foods and performing sedentary activities. They
are encouraged to learn how to recognize lack of control on food intake under stressful
or anxious situations and how to exercise self-control.

Recommendations on the use of tobacco

The PREDIMED-PLUS dietitians make no recommendations on the use of tobacco. This
is the responsibility of the medical professionals in the primary care centers in
accordance with usual medical practice.

Individual motivational interviews

Personal interviews with the dietitian at each individual visit are adapted to the
participant’s clinical conditions, preferences and beliefs. Dietary changes are introduced
in order to achieve the recommended diet for each participant and suitable lifestyle
changes are incorporated. Objectives are accorded via a negotiated agreement between
the two parties (dietitian and participant) depending on what participants consider to be
an attainable goal. The main objective is to change not only the participant’s consumption
of certain foods but also his or her overall dietary pattern. Attention can vary between
changing portion sizes, changing the frequency of dietary components, and changing
cooking methods.
Achievements made in the previous months, however minor, are always considered
an essential support mechanism for improving self-esteem and self-reward. Special care

43
is taken to ensure that participants do not receive contradictory dietary advice from health
professionals external to the PREDIMED-PLUS trial.
As described, each participant receives oral and written information on the food
components and culinary customs of the energy–restricted Mediterranean diet, as
well as charts for self-registering and self-monitoring changes in weight and waist
circumference at each visit.
Participants who during the active weight-loss phase have observed a lower weight
loss than expected or who have not maintained the weight loss they had achieved
receive special reinforcement and a series of rescue measures to help them achieve
weight loss and weight-loss maintenance. In such cases, agreements are negotiated
between the dietitians and the participants.

Role of the dietitians

The PREDIMED-PLUS dietitians are directly responsible for the dietary intervention.
They have been specifically trained and certified to deliver the PREDIMED-PLUS
intervention protocol. All intervention procedures are conducted in accordance with the
PREDIMED-PLUS operation’s manual. Throughout the study, annual meetings will be
held at which the dietitians will discuss any problems they may have identified and find
possible solutions. The dietitians and trial coordinators will discuss any problems arising
during the trial, thus ensuring a process of continuous feedback.

12. Training and calibration procedures

A general trial operations manual and staff training documents are set forth to ensure
standardized procedures across the various recruiting centers. Before implementation of
the protocol, study personnel attended a 3-day training course at the coordinating center.
This included theoretical and practical group discussions with experts on lifestyle
interventions in order to convey the goals of the study, develop all the specific aspects
involved in implementing the intervention, and impart training on the informed-consent
process, anthropometric and blood pressure measurements, data collection by optical
scanning or online systems, and biological sample collection and processing. The
abilities of all contracted personnel were evaluated at personal interviews during this
training course. The research team stressed the importance of creating a trusting and
empathic relationship with the participants and paying attention to their individual needs
in order to maximize their motivation and retention into the trial. Study personnel keep a
copy of the operations manual detailing all the training points. In addition, all the
personnel responsible for the intervention are attending annual meetings and are in

44
constant contact with the principal investigators in order to ensure standardized
implementation of the trial protocol. In accordance with the protocol, all scales and other
measurement instruments are periodically calibrated.

13. Retention and compliance with strategies and supervision procedures

The recruitment of participants and the compliance rates for the intervention
strategies are crucial to the success of a trial of this nature. For this reason, a run-in
(pre-evaluation) period prior to randomization was planned: only participants who
adhere to all the requirements of the protocol during the run-in period are accepted into
the trial. The lag time between the end of the run-in period and the start of the intervention
ranges from one week to one month.
The researchers involved in this trial have already gained invaluable experience in
managing long-term trials through the PREDIMED trial, wherein they developed
strategies for ensuring participants’ compliance with the protocol and encouraging their
long-term retention. We understand that this is a particularly sensitive aspect for
participants in the control group. Therefore, at each group session of both the intensive
intervention group and the usual care group, virgin olive oil and mixed nuts is provided
at no cost to all participants. Our experience in the PREDIMED trial showed that such
gifts, especially the virgin olive oil, greatly helped to encourage participants’ retention.
Other retention strategies include providing feedback on findings during follow-up to the
participants’ usual health-care providers as well as supplying other non-coercive material
incentives for both groups. Additionally, in the intensive intervention group, where
significant weight-loss is anticipated, contact with participants is ongoing and flexible
interventions and rescue measures tailored to the participants’ needs are implemented.
Self-control, self-reward and self-monitoring techniques are also reinforce participants’
compliance with the intervention. Finally, the intervention is adapted to the needs of the
participants, which should encourage compliance.

14. Biological samples and laboratory procedures

The nursing staff contracted at each recruiting center is responsible for collecting,
processing and storing the biological samples in freezers at a temperature of -80ºC.
Blood samples are collected at the recruiting centers in the same way as they are
collected at the participants’ usual healthcare centers. In addition, 55.5 ml of blood are
extracted and collected in the following tubes: two 10 ml K2E EDTA tubes; one 4.5 ml
citrate tube; and two 10 ml and 6 ml gel serum separator tubes. The serum, citrate
plasma and EDTA plasma samples are distributed in aliquots of 200 μl and 500 μl and

45
stored at -80°C for future analyses at the recruiting centers. For the intensive intervention
group, the biochemical measurements are performed in a blind fashion and in the same
batch for consecutive samples of each participant. Each recruiting center has an ultra-
low-temperature freezer with enough capacity to store biological samples until final
delivery. All biological samples are processed at each recruiting center no later than one
hour after extraction. During transportation from the primary care centers to the
laboratories, the biological samples are stored at 4ºC in a portable cooler. Urine tests
are conducted at the recruiting centers in the same way they are conducted at the
participants’ usual healthcare centers in accordance with the specific PREDIMED-PLUS
protocol for collecting biological samples.

15. Quality control

The general database for the PREDIMED-PLUS trial is managed and maintained by the
research group of the IMIM Institute (CIBERobn). The food-frequency questionnaires
and the food records, as well as quality of life test, clok and Minimental tests are
processed and managed at the University of Navarra. Data collected from
accelerometers to measure physical activity are processed at the Malaga recruiting
center in collaboration with the CSIC/UAM. These data are sent every three months to
the IMIM, where they are incorporated into the General Database. Event detection data,
collected from information gathered during the intervention and at the follow-up visits,
are introduced into specific forms at the recruiting centers, preferably using online
systems, and sent at least once a month to the data manager at the IMIM, who sends
monthly reports of missing or inappropriate entries back to the recruiting center
coordinators to solve any raised queries. The IMIM also sends monthly reports to the
different recruitment centers with the sumsample of each node. The steering committee
has been set up to ensure the quality of the project and correct any flaws or divergences.
This committee was made up of Jordi Salas-Salvadó (PREDIMED-Plus coordinator),
Miguel Angel Martínez-González, Ramón Estruch, Montserrat Fitó, Emilio Ros, and
Dolores Corella. In december 2016, Dr. Francisco Tinahones (Hospital de Málaga) was
incorporated as the seventh member of the Steering Committee.
At every annual PREDIMED-PLUS meeting, the IMIM conduct a current data
management information session. An annual summary is sent to the recruiting center
coordinator for distribution to all groups.
To reduce data entry expenses and speed up processing, the questionnaires and
data forms are processed by optical scanning or by online data transfer forms. The data
forms are entered in duplicate and missing data checks are performed. All forms sent to

46
another recruiting center must be photocopied and stored at that center. After data entry,
cross-form edit checks are performed and any data inconsistencies are identified. To
detect any still-unsolved problems, audits are run periodically at each recruiting center.
Reports are drafted to summarize any problems in the database and provide an
additional step to ensure the quality and accuracy of the data. To minimize the possibility
of error, a detailed operations manual has been prepared.
Annual staff training meetings are conducted. The data manager and an audit
committee evaluate the performance of each recruiting center. Appropriate new
procedures and corrective measures are implemented whenever deficiencies are noted.
Until the end of the trial, all field centers are masked to the trial outcome data except for
the two trial statisticians, one in Navarra (M.A. Martínez-González) and one at the IMIM
in Barcelona (Joan Vila), who will always perform the statistical analyses in duplicate
with two statistical analysis units. Because of the nature of the trial, however, the
dietitians at each field center know which intervention has been assigned to each
participant. The medical doctors who prepare the annual report on the ad hoc review of
the participants’ medical records are blinded to group assignment, as the Clinical Event
Ascertainment Committee. The members of the Steering Committee, who attend the
meetings of the Data and Safety Monitoring Board, also remain blinded to the results of
intermediate analyses throughout the trial. The Steering Committee is informed of the
total number of events observed but not of the groups in which they occurred.

STATISTICAL ANALYSIS PLAN

All analyses will be performed on an intention-to-treat basis. Miguel A. Martínez-
González will be the senior statistician responsible for the statistical analysis plan. All
major data analyses will be conducted under his supervision. Statistical analyses for the
main aims of the study will be also conducted in duplicate by the center at IMIM,
Barcelona (responsible statistician at IMIM: Joan Vila).

1. Analysis of the effect of the intervention

Since the data take into account time to the event, Cox’s regression models will be
used to determine the effect of the intervention on the incidence of cardiovascular events.
For changes in weight and waist circumference, mixed models of analysis of variance
and generalized estimating equations (GEE) will be used. These models will include the
following adjustment covariates:
1) All factors that, according to the scientific literature, are related to the event; and
2) All factors that reach statistical significance in univariate analyses.

47
In these models we will evaluate: 1) the proportional hazards assumption; 2) the linearity
of the continuous variables, using smoothing spline methods; 3) the effect of extreme
observations on the estimation of parameters, by calculating delta-beta values. The use
of further approaches (i.e. normalizing transformations, stratified analyses, etc.) will
depend on the results obtained above. Given that participants will be clustered by
recruiting centers, some degree of correlation structure may be expected. Center will
therefore be included as a stratification variable, including frailty estimates, in the Cox
regression models. The goodness-of-fit of the models will be examined using the
modified Hosmer-Lemeshow test for survival studies. Robust estimators of variance that
account for the clustering effect of members of the same household (the second member
is not randomized for feasibility reasons) will be used to take into account the intra-cluster
correlation. Sensitivity analyses will be conducted after excluding the second (non-
randomized) members of the same household. In addition to the stratification by center,
all Cox models will be also stratified by sex and educational level.

2. Interim analyses and stopping rules

Data from the PREDIMED-PLUS trial will be analyzed after 3 years of median follow-up,
after 5 years of median follow-up, and at the end of the trial. For methodological reasons
but especially for ethical ones, suitable follow-up for a trial must include at least one
interim analysis (Schulz, Grimes, 2005). However, to preserve an overall alpha error of
0.05, interim analyses have to be penalized. We will use the O’Brien and Fleming
boundaries (O'Brien and Fleming, 1979). With this method, the boundaries are stricter at
the earlier stages of the study than at the later ones. Applying this rule leads to the
following p values for stopping the trial:
First interim analysis (median follow-up: 3 years); threshold p value: 0.0005.
Second interim analysis (median follow-up: 5 years); p value: 0.014.
Final analysis (median follow-up: 8 years); p value: 0.045

These p values should not be considered compulsory for stopping the trial but guidelines
for guaranteeing the security of the data. In making their decision, the Data and Safety
Monitoring Board must take into account, for example, the size of the effect, the follow-
up time at each recruiting center, the heterogeneity between the effects at the recruiting
centers, as well as evidence from other current trials and observational studies. All the
above must be taken into consideration when deciding either to continue or to interrupt
the trial after each interim analysis. Reasons for interrupting the trial include: 1)

48
convincing evidence of the beneficial effect of the intervention (the trial will be stopped
only if the effect of the intervention is great); 2) convincing evidence of a harmful effect
from the intervention; 3) results suggesting it is highly unlikely that the proposed
hypothesis will be accepted due to, for example, a very small effect of the intervention
that dramatically affects the trial’s statistical power.

3. Estimations of sample size

We will determine the effect of the intensive weight-loss lifestyle intervention with an
energy-restricted Mediterranean diet on the two primary outcomes below, assuming a
two-tailed alpha error of 0.05.

1. Effect of the intervention on the incident CVD (non-fatal myocardial infarction, non-
fatal stroke, and cardiovascular death). The cumulative projected incidence after
including as primary events all non-fatal acute myocardial infarctions and all
microinfarctions with positive high-sensitivity troponin tests after 6 years will be at least
10% in the control group, if we take into account the results of the PREDIMED trial after
4.8 years (which did not include high-sensitivity troponin tests). The hazard ratio (HR) for
the combined primary endpoint is anticipated to be 0.70 (Estruch et al., 2013) and will
probably be even lower (greater protective effect) if we consider that in the PREDIMED
trial no energy restriction was implemented, physical activity was not encouraged, and
weight loss was not a target of the intervention. Under these assumptions, therefore,
even if the dropout rates were to reach 20%, the required sample size would be 2,400
per group (see Figure 3). To be conservative, initially we aim to recruit 6,000 participants
and assign 3,000 participants to each group. However, the final sample included 6,874
participants distributed in 23 centers, 3,406 participants assigned to the intensive
intervention group and 3,468 to the control group.

49
FIGURE 3. Estimation of the sample size required per intervention group in the
PREDIMED-PLUS trial

2. Effect of the intervention on weight change. Based on previous studies, we can

expect a minimum weight change for participants in the control group and a weight loss
of 3-4.5 kg for those in the intensive lifestyle intervention group, with a standard deviation
of 8 kg (Shai et al., 2008; Sacks et al., 2009; Wing, 2010). If we assume our intervention
will have only a small effect on weight change and then calculate sample size according
to a weight change of 1 kg in the usual care group, a weight change of 3 kg in the
intensive lifestyle intervention group, and a standard deviation of 8 kg, in order to achieve
a statistical power of 0.80 we would need a sample size of only 337 in each group. Since
the number of participants recruited is much higher than this figure, the statistical power
needed to reach this objective is largely guaranteed.

STRENGTHS AND LIMITATIONS

1. Strengths
A. This trial provides a multidisciplinary approach to tackling the serious problem
presented by the overweight and obesity epidemic. Our target group comprises obese
or overweight adults, who represent an increasing proportion of the general population.
For these subjects, an intervention based on a profound lifestyle change incorporating
improvements in the dietary pattern, weight loss, behavioral therapy, and increased
physical activity can be a novel and useful model for reducing the burden of obesity and
associated diseases, thus contributing to the sustainability of the healthcare system. The
trial clearly addresses priority objectives of the public healthcare system since it tackles
both the principal epidemic of our times (overweight and obesity) and the principal cause
of death around the world (CVD).

50
B. This innovative proposal presents a novel paradigm for nutritional recommendations
aimed at achieving weight loss, i.e., a traditional dietary pattern characterized by a
moderate-to-high fat content. We believe this new approach will help improve
compliance with the intervention and overcome the main challenge of any dietary
interventions aimed at fighting overweight and obesity: long-term weight loss
maintenance (Shai et al., 2008; Beunza et al., 2010; Romaguera et al., 2010).
C. The intervention is well structured and the trial is suitably designed for determining
the effect of the intervention on the main clinical outcomes. Moreover, since the trial is
conducted in the context of primary healthcare and incorporates epidemiological, clinical
and basic aspects, it has a high capacity for both transferability and reproducibility.
D. The research team includes investigators with invaluable experience in lifestyle
intervention trials (e.g., PREDIMED). As these investigators come from a wide range of
fields, their work will be complementary and the trial’s chances of success will be
enhanced. All these reasons, together with the success and achievements of the
PREDIMED trial, attest to the viability of this trial proposal.
E. From a strategic perspective, this is a timely proposal since it provides continuity for
the collaborative project in which most CIBEROBN centers have participated, i.e.,
PREDIMED.

2. Limitations
A. Our study enrolled participants aged between 55 and 75 years old. This may preclude
generalization to younger age groups.
B. In a large-scale clinical trial, one limitation to consider is participants’ dropout rates.
However, we hope to ensure compliance in both groups by: a) providing free foods (olive
oil and mixed nuts); b) establishing personal relationships with each participant via
individual and group sessions; c) administering, at the start of the study, the Prochaska
and DiClemente Stages of Change Model, by which a low predicted probability of
changing dietary habits will be a criterion for exclusion; and d) establishing, at the start
of the study, a one-month run-in period in order to identify and select participants with a
greater likelihood of compliance with the protocol and retention into the study (see
section 5 and 13).
C. Homogeneity of the interventions is difficult because it is based on three components:
diet, physical exercise, and behavior. For this reason we have developed a detailed
protocol for implementing the intervention and have established a committee for each
intervention component. We also conducted a staff training session at the beginning of
the study and will conduct annual follow-up sessions throughout.

51
COMMITTEES AND GOVERNANCE
The PREDIMED-PLUS Executive Committee includes the principal investigators
from all the participating centers (see Annex 1). It provides scientific and strategic
orientation for decision-making and is responsible for designing, implementing and
publishing the study’s protocol and guaranteeing the quality of its implementation and
management. It determines its own guidelines and approve the criteria and guidelines of
the other committees within the study. It convene at least twice a year to discuss and
report on the study’s progress.
In February 2018, Dr. Julia Wärnberg (School of Health Sciences) replaced the
principal investigator of the recruitment center of the University of Malaga, Prof. Enrique
Gómez-Gracia, who continues in the trial as a principal investigator of a new support
group (A8).

The Steering Committee, made up of Jordi Salas Salvadó (Chair), Miguel Angel
Martínez-González (PI of the ERC-Advanced Research Grant), Ramón Estruch,
Montserrat Fitó, Emilio Ros, Dolores Corella and Francisco Tinahones who was
incorporated as the seventh member of the Steering Committee in December 2016, is
responsible for ensuring the quality of the project and correcting any flaws or divergences
that may be detected.

Data Safety and Monitoring Board

To ensure the smooth running of the trial and the safety of participants, an
Independent Data Safety and Monitoring Board has been set up. This Board is made
up of: Chairman, Meir J. Stampfer (Harvard School of Public Health); members Joan
Sabaté (Loma Linda University), Arne Astrup (Copenhagen University), Francisco
Fernandez-Avilés (Universidad Complutense of Madrid) and Xavier Pi-Sunyer (Columbia
University). The Board has its first meeting in 2015 and is convened at least once a year
to review the implementation of the protocol and monitor the trial’s progress. It examines
the competence of each recruiting center, evaluate their compliance with the study’s
objectives, and decide whether they may continue in the trial.
In addition, a report is mailed periodically by the PREDIMED-PLUS Steering Committee
to the Board members with relevant statistical analyses for judging on the continuation
of the PREDIMED-PLUS trial. Throughout the study, the Board members can request
any statistical analysis on a blinded or unblinded basis. The Board may recommend
termination of the trial at any time if an unacceptable incidence of adverse events or

52
significant differences in mortality between study groups are observed. The Executive
Committee of the PREDIMED-PLUS trial, however, will make the final decision.

SOURCES OF FUNDING AND ADMINISTRATIVE ISSUES

This study was supported by the official funding agency for biomedical research of the
Spanish government, Instituto de Salud Carlos III (ISCIII), through the Fondo de
Investigación Sanitaria (FIS) that awarded us four coordinated research projects for the
2014-2016 and 2017-2019 annual periods (IP of the coordinated projects Jordi Salas-
Salvadó), and for the annual periods of 2015-2017 and 2018- 2020 (IP of the coordinated
projects Josep Vidal) ; CIBEROBN, which is co-funded by the European Regional
Development Fund, and by the European Research Council (Advanced Research Grant
2013-2018; 340918) grant to Miguel Ángel Martínez-Gonzalez.

Trial’s website
http://www.predimedplus.com

Contact’s name and address

Prof. Jordi Salas-Salvadó, MD, PhD. Human Nutrition Unit. Faculty of Medicine and
Health Sciences, Universitat Rovira i Virgili, C/ Sant Llorenç, 21, 43201 Reus (Spain).
Telephone number: +34 977759312; Fax number: +34 977759322. E-mail:
jordi.salas@urv.cat.
Prof. Miguel A. Martínez-González, MD, PhD, MPH, Department of Preventive
Medicine and Public Health, Facultad de Medicina–Clínica Universidad de Navarra,
Irunlarrea 1, 31008 Pamplona, Spain. Phone: +34 636 355 333, E-mail:
mamartinez@unav.es

PREDIMED-PLUS Registration
The PREDIMED--Plus trial was registered at the International Standard Randomized
Controlled Trial (ISRCT; http://www.isrctn.com/ISRCTN89898870) with number
89898870 and a registration date of 24 July 2014.

53
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ANNEX 1. SUB-STUDIES

1. Body composition
DEXA or computed tomography is used to measure body composition at recruiting
centers wherein the necessary equipment and technology are available. Body
composition is analyzed by General Electric Lunar DEXA scanner at the Rovira i Virgili
University, Universitat de les Illes Balears, Hospital Clinico de Barcelona, and the
Departments of Preventive Medicine and Nutrition in Navarra.

2. Other sub-studies
Depending on available funds, sub-studies will be conducted to evaluate gene
environment interactions, epigenetic factors such as DNA methylation, histone
modification and microRNA alterations, the composition and function of intestinal
microbiota by pyrosequencing, and the effect of the intervention on metabolomics,
transcriptomics and proteomics.

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ANNEX 2. PARTICIPATING CENTERS

Recruitment centers
ID PI Email address Center
Escuela de Ciencias de la Salud,
01 Julia Wärnberg jwarnberg@uma.es
Universidad de Málaga-IBIMA, Málaga
Distrito Sanitario de Atención Primaria
02 José Lapetra jlapetra@ono.com
de Sevilla, Sevilla
Universidad de Navarra - Nutrition,
03 J. Alfredo Martínez jalfmtz@unav.es
Pamplona
María Adoración mariaadoracion.romaguera@
04 Romaguera; Hospital Son Espases, Palma
Miquel Fiol ssib.es; miquelfiol@yahoo.es
Hospital Clínic-Medicina Interna,
05 Ramon Estruch restruch@clinic.ub.es
Barcelona
Institut Hospital del Mar
06 Montserrat Fitó mfito@imim.es
d’Investigacions Mèdiques, Barcelona
07 Jordi Salas-Salvadó jordi.salas@urv.cat Universitat Rovira i Virgili, Reus
08 Aurora Bueno abueno@ugr.es Universidad de Granada, Granada
09 Clotilde Vázquez cvazquezma@gmail.com Hospital Ramón y Cajal, Madrid
Miguel Ángel Universidad de Navarra -
10 mamartinez@unav.es
Martínez-González Epidemiology, Pamplona
11 Fernando Aròs aborau@htxa.osakidetza.net Hospital Universitario Araba, Vitoria
12 Dolores Corella dolores.corella@uv.es Universidad de Valencia, Valencia
Universidad de las Palmas de Gran
13 Lluis Serra-Majem lserra@dcc.ulpgc.es
Canaria, Las Palmas
Hospital de Bellvitge, L’Hospitalet de
14 Xavier Pintó xpinto@csub.scs.es
Llobregat, Barcelona
15 José López Miranda jlopezmir@gmail.com Universidad de Córdoba, Córdoba
16 José Maria Ordovás Jose.Ordovas@tufts.edu IMDEA, Madrid
17 Pilar Matía Martín pilar.matia@gmail.com Hospital Clínico, Madrid
Francisco
18 fjtinahones@hotmail.com Hospital de Málaga, Málaga
Tinahones
19 Josep Tur pep.tur@uib.es
Universitat de les Illes Balears, Palma
Hospital Clínic – Endocrinología,
20 Josep Vidal JOVIDAL@clinic.ub.es
Barcelona
Universidad Miguel Hernández, Sant
21 Jesús Vioque López vioque@umh.es
Joan d'Alacant, Alicante
Miguel Delgado
22 mdelgado@ujaen.es Universidad de Jaén, Jaén
Rodríguez
23 Vicente Martin vicente.martin@unileon.es Universidad de León, León

70
Support centers

ID PI Email address Center

A1 Emilio Ros eros@clinic.ub.es Hospital Clínic, Barcelona
Rosa M. Lamuela-
A2 lamuela@ub.edu Universitat de Barcelona
Raventós
A3 Guillermo Sáez guillermo.Saez@uv.es Universitat de Valencia
María del Puy
A4 mariapuy.portillo@ehu.es Universidad del País Vasco
Portillo
A5 Ascensión Marcos amarcos@ictan.csic.es CSIC, Madrid
Universitat Jaume I de
A6 Cristina Botella botella@uji.es
Castellón
Fernando Hospital Universitari de
A7 ffernandez@bellvitgehospital.cat
Fernández-Aranda Bellvitge
Medicina Preventiva, Facultad
Enrique Gómez- egomezgracia@gmail.com
A8 de Medicina, Universidad de
Gracia
Málaga, Málaga

71