Journal of

Clinical Medicine

Article
Whole-Body Cryotherapy Is an Effective Method of
Reducing Abdominal Obesity in Menopausal Women
with Metabolic Syndrome
Magdalena Wiecek 1, * , Jadwiga Szymura 2 , Justyna Sproull 3 and Zbigniew Szygula 4
1 Department of Physiology and Biochemistry, Faculty of Physical Education and Sport, University of Physical
Education in Krakow, 31-571 Kraków, Poland
2 Department of Clinical Rehabilitation, Faculty of Motor Rehabilitation, University of Physical Education in
Krakow, 31-571 Kraków, Poland; jadwiga.szymura@awf.krakow.pl
3 Ph.D. Studies, Faculty of Physical Education and Sport, University of Physical Education in Krakow,
31-571 Kraków, Poland; bednarekjustyna1@gmail.com
4 Institute of Biomedical Sciences, Faculty of Physical Education and Sport, University of Physical Education
in Krakow, 31-571 Kraków, Poland; wfszygul@cyf-kr.edu.pl
* Correspondence: magdalena.wiecek@awf.krakow.pl; Tel.: +48-507857329




Received: 31 July 2020; Accepted: 28 August 2020; Published: 30 August 2020


Abstract: Abdominal obesity predominates in menopausal women (MW) and contributes to the
development of metabolic syndrome (MetS). It is associated with increased mortality related to
cardiovascular disease, diabetes and fatty liver disease. The effects of whole-body cryotherapy (WBC)
on body composition and the blood concentration of irisin, interleukin-6 (IL-6) and C-reactive proteins
(CRP) in MW with MetS and in healthy women (HW), were assessed. The study included 19 women
with MetS (61.53 ± 3.99 y, BMI 30.09 ± 4.98 kg/m2 ) and 18 HW (60.28 ± 3.63 y, BMI 25.50 ± 2.37 kg/m2 )
who were subjected to 20 WBC treatments at −130 ◦ C for 3 min daily. In both groups, body mass (BM),
BMI, abdominal circumference, triceps skinfold, total fat mass and percentage of leg fat significantly
decreased after 20 WBC sessions. Additionally, the percentage of total, trunk and android fat in
the MetS group were significantly decreased after 20 WBC applications. Waist circumference (WC)
and waist-to-height ratio (WHtR) significantly decreased in both groups, and in the HW group,
hip circumference and abdominal skinfold also significantly decreased after 10 WBC and 20 WBC
treatments. In both groups, the concentration of plasma irisin significantly increased after 1 WBC
and 10 WBC exposures, while the concentration of IL-6 significantly increased only in MetS group
after 10 WBC and 20 WBC, and were significantly higher than in HW. CRP concentrations were
significantly higher in the MetS group than in HW before 1 WBC, after 1 WBC and 10 WBC sessions,
but not after 20. In the MetS group, there were significant negative correlations between the change
in irisin level and the changes in WC and BM, and between the level of irisin and the change in
percentage of total fat, and significant negative correlations between the change in IL-6 level and
changes in WC, waist-to-hip ratio and WHtR. Whole-body cryotherapy, assuming the application
of 20 treatments in the series, reduces abdominal obesity in menopausal women indirectly through
the secretion of irisin and IL-6, and can be used as adjunctive therapy in the treatment of metabolic
syndrome. Our conclusion is limited to menopausal women with low–moderate physical activity for
whom its level as well as diet were not changed during the treatment.

Keywords: whole-body cryotherapy; body composition; visceral obesity; menopausal women;

metabolic syndrome; irisin; interleukin-6

J. Clin. Med. 2020, 9, 2797; doi:10.3390/jcm9092797 www.mdpi.com/journal/jcm

J. Clin. Med. 2020, 9, 2797 2 of 21

1. Introduction
Obesity, defined as abnormal or excessive fat accumulation that presents a risk to health, is a
serious global problem [1]. Overweightness and obesity are more common in women [2,3]. Globally,
the prevalence of overweightness (BMI ≥ 25 kg/m2 ) and obesity (BMI ≥ 30 kg/m2 in adults has increased
among men from 25.4% in 1980 to 38.5% and 10.1% (respectively) in 2015, while among women, from
27.8% and 8.9% to 39.4% and 14.8%, respectively [3]. It has been estimated that by 2025, the percentage
of obese males world-wide will total 18%, and for women, 21% [2]. The highest percentage of obese
women occurs in the menopausal period [3]. Menopausal women are most often characterised by
abdominal obesity, also known as android or visceral obesity [4,5]. In menopausal women, the increase
in BMI and total body mass, which consists of fat mass and the remaining tissues (lean body mass),
is mainly due to an increase of fat in the abdominal area. In this period, the amount of visceral fat
among women increases by over 40%, and the amount of subcutaneous fat in the abdominal region by
about 20% [6]. In population studies carried out in the USA and Poland, it has been shown that over
60% of menopausal women were characterised by abdominal obesity [7,8]. Abdominal obesity is a
major cause of cardiovascular disease, diabetes, musculoskeletal disorders, especially osteoarthritis,
and some cancers (including endometrial, breast, ovarian, prostate, liver, gallbladder, kidney, and
colon) [1].
Abdominal obesity contributes to the development of metabolic syndrome (MetS) [9], in which
dyslipidemia, hyperglycaemia and arterial hypertension are also diagnosed [10–13]. It is estimated that
the occurrence of MetS in Europe, although varying in different countries, affects about 37.6% of women
aged 60–78, while in men, this value is 21.8% [14,15]. In Poland, MetS is present in approximately 46% of
women and 34% of men aged 60–74 [16]. The most common diagnostic criterion was abdominal obesity,
which was more common in women than in men, in 40.0% and 29.1% of people, respectively [17].
Abdominal obesity leads to an imbalance in the production and secretion of pro-inflammatory
(IL-6, IL-1β, TNF-α) and anti-inflammatory (IL-10) factors, and contributes to the occurrence of chronic,
low-grade systemic inflammation [18,19]. The concentration of acute phase inflammatory protein like
C-reactive protein (CRP) and markers of oxidative damage is higher in obese people and positively
correlates with BMI, percentage of adipose tissue and triglyceride levels [20].
The epidemic nature of obesity and its consequences is a major worldwide, public health
problem [14–17,21–23]. There are studies on the effectiveness of using whole-body cryotherapy as a
non-pharmacological method of supporting the treatment of obesity and metabolic disorders [24–28].
Whole-body cryotherapy (WBC) consists in short-term (1–3 min), repeated exposure to low
temperatures (from −110 ◦ C to −160 ◦ C) on the largest possible surface of exposed skin [29]. It has been
shown that WBC treatments cause a generalised physiological response of the body [30]. According to
the International Classification of Medical Procedures (ICD-9), WBC is a physiotherapeutic treatment
(93.3950). In Poland, WBC treatments belong to medical services used as treatments preceding
kinesiotherapy with analgesic, anti-swelling and anti-inflammatory effects, mainly in rheumatoid and
neurological diseases. As treatments reimbursed by the National Health Fund (13/2019/DSOZ) [31],
they can be applied 6 months apart, each in the number of 10 treatments, daily on working days in 2
consecutive series. In people qualified for the procedures, there are no contraindications for using
more WBCs in a series and their more frequent repetition. In scientific research, WBC is most often
used once a day, applying a total of 5 to 30 treatments [29].
Depending on the number of treatments, duration, frequency and temperature range, various
metabolic effects of these treatments are possible [30]. There is a need to study the distant effects of WBC
application. To our knowledge, only Szymura et al. [32] showed an increase in IL-3 and erythropoietin
levels 7 days after the completion of a series of 24 WBC treatments in older marathon runners and
non-trained men. Although not all research results are consistent, it has been demonstrated that WBC
may have a beneficial effect on hormonal and metabolic changes, and is also anti-inflammatory [29,30].
An increase, inter alia, has been noted in the concentration of IL-6, without changes in the concentration
of TNF-α in the blood after WBC procedures [33], which indicates an anti-inflammatory effect and
J. Clin. Med. 2020, 9, 2797 3 of 21

may, via IL-6, positively influence metabolism [34,35]. Improvement in lipid profile [24,25] and a
decrease in the concentration of resistin, visfatin [24] and asprosin [27] were found in the blood of
obese individuals as a result of WBC application. However, there was no effect of WBC on changes in
leptin and adiponectin levels [24,27]. Obese men characterised by low physical activity showed an
increase in irisin concentration as a result of exposure to cryogenic temperatures [26].
Irisin is a 29-amino acid fragment of the extracellular domain of the FNDC5 protein, the gene of
which is expressed mainly in myocytes [36] and adipocytes [37]. A negative correlation was found
between the concentration of irisin in the blood and age [38]. Irisin affects the browning of white adipose
tissue; by inducing UCP-1 expression, it increases energy expenditure as a result of non-shivering
thermogenesis; influences glucose homeostasis and improves insulin sensitivity [39]. In an animal
model, a protective role of irisin against obesity has been demonstrated by reducing the concentrations
of LDL, TG, glucose and leptin in the blood [40]. People with MetS demonstrated lower levels of irisin
and, at the same time, higher levels of CRP and IL-6 in the blood compared to individuals without
MetS. Lower levels of irisin were found in people with high CRP, but not in those with high IL-6 [41].
It is known that physical exercise is a factor increasing irisin secretion in obese people [42]. In
two earlier conducted studies, it was indicated that exposure to cryogenic temperatures may also be a
factor inducing the secretion of this hormone, although the studies were carried out only in men, both
with normal body mass [43] and those obese [26].
The aim of our study is to evaluate the effect of 10 and 20 WBC sessions performed daily
in series of 5 on irisin secretion and body composition in menopausal women with and without
metabolic syndrome.
Taking into account the metabolic effect of irisin and the induction of the effect of cryogenic
temperatures on the secretion of this hormone in obese men, as shown in previous studies [26], and
the induction of IL-6 secretion, without intensifying inflammation [33], we hypothesise that 20 WBC
treatments reduce the content of adipose tissue and abdominal obesity in menopausal women, which
is indirectly associated with an increase in the concentration of irisin and IL-6 in the blood.

2. Methods

2.1. Study Design

As in the previous study [27], Caucasian menopausal women (no menstruation for at least 12
months) aged 55–70 years, who had no contraindications for the application of WBC, were included
in the study [29]. People who had participated in WBC procedures within the last 6 months, as well
as smokers, alcohol abusers, chronically ill patients, those taking medications on a permanent basis,
except for antihypertensive therapy, people whose diet deviated from the standards developed for the
Polish population by the Institute of Food and Nutrition [44], e.g., following specific diets (vegetarian,
vegan, diabetic, low-calorie) or dietary supplements that may affect the metabolic rate, were also
excluded from the trial.
The first stage of the study included internal medicine examination, during which the presence of
metabolic syndrome was diagnosed, taking into account the criteria proposed by the National
Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATP III) [10–13] as well as
cardiological and gynaecological examinations.
MetS was diagnosed in people who met at least 3 of any of the following criteria:

• Waist circumference > 88 cm;

• TG concentration ≥ 150 mg/dL;
• HDL concentration < 50 mg/dL;
• fasting glucose ≥ 100 mg/dL; and
• systolic blood pressure (SBP) ≥ 130 mmHg o diastolic blood pressure (DBP) ≥ 85 mmHg or anti
hypersensitive therapy.
J. Clin. Med. 2020, 9, 2797 4 of 21

During the second stage, a series of 20 WBC treatments was performed. Somatic measurements
(body mass, waist, abdominal and hip circumferences, skin fold thickness), body composition
assessment and biochemical determinations (irisin, IL-6, CRP) were performed in fasting state before
the 1st WBC, the day after the 10th WBC and after the 20th WBC treatments. Biochemical determinations
were also performed the day after WBC 1.
Physical activity and diet were evaluated. The subjects were asked to maintain their current
physical activity and diet, and not to undergo any biological regeneration treatments (i.e., massage,
sauna, hydrotherapy) while using WBC.
The study was conducted in accordance with the Declaration of Helsinki. The methodology of the
study was approved by the Bioethical Committee of the Regional Medical Chamber (96/KBL/OIL/2015,
3 July 2015).

2.2. Participants
The inclusion criteria were met by 41 out of the 79 volunteers, and 20 were diagnosed with
MetS. During the period of using WBC treatments, 4 people resigned from the study, including 2 for
organisational and 2 for health reasons (upper respiratory tract infection and extensive cut of the facial
skin as a result of a fall not related to the use of WBC). The entire study programme was completed
by 37 volunteers, 19 of whom had MetS, the remaining women were healthy and controlled. In the
study group, 7 women exhibited normal body mass (BMI 23.4 ± 2.29 kg/m2 ), 22 were overweight
(27.48 ± 2.51 kg/m2 ) while 8 were obese (BMI 32.76 ± 6.05 kg/m2 ). The characteristics of the participants,
according to the MetS and healthy division, are presented in Table 1.

Table 1. Age and medical qualification.

Variable Metabolic Syndrome Healthy Total Min-Max

Age (years) 61.53 ± 3.99 60.28 ± 3.63 60.92 ± 3.82 55.00–67.00
Erythrocytes (106 /µL) 4.62 ± 0.22 4.59 ± 0.24 4.61 ± 0.23 4.20–5.10
Haemoglobin (g/dL) 13.89 ± 0.55 13.94 ± 0.55 13.92 ± 0.55 13.20–15.30
Haematocrit (%) 40.82 ± 1.75 41.14 ± 1.62 40.98 ± 1.67 38.60–44.60
ESR (mm/h) 19.58 ± 10.67 16.11 ± 9.65 17.89 ± 10.19 2.00–43.00
Platelets (103 /µL) 246.53 ± 77.69 254.89 ± 56.59 250.59 ± 67.44 61.00–457.00
Leukocytes (103 /µL) 6.16 ± 1.25 * 5.29 ± 1.06 5.74 ± 1.23 3.82–8.53
Neutrophils (103 /µL) 3.04 ± 0.86 2.60 ± 0.57 2.83 ± 0.76 1.60–5.40
Lymphocytes (103 /µL) 2.24 ± 0.56 1.92 ± 0.50 2.08 ± 0.55 1.10–3.40
Monocytes (103 /µL) 0.48 ± 0.11 0.50 ± 0.14 0.49 ± 0.12 0.26–0.84
Eosinophils (103 /µL) 0.18 ± 0.09 0.17 ± 0.09 0.18 ± 0.09 0.06–0.42
Basophils (103 /µL) 0.02 ± 0.03 0.04 ± 0.04 0.03 ± 0.03 0.00–0.10
HbA1C (%) 5.84 ± 0.28 * 5.67 ± 0.29 5.75 ± 0.29 5.20–6.50
Insulin (µIU/mL) 12.12 ± 5.65 * 8.27 ± 2.50 10.25 ± 4.77 4.40–28.20
HOMA-IR 3.03 ± 1.52 * 1.92 ± 0.61 2.49 ± 1.29 0.95–7.19
TCHOL (mg/dL) 213.50 ± 37.57 223.56 ± 35.55 218.39 ± 6.45 157.90–295.28
LDL (mg/dL) 127.78 ± 37.13 137.81 ± 35.04 132.66 ± 35.99 57.13–214.12
AIP (log10 TG/HDL) 0.38 ± 0.23 * 0.22 ± 0.20 0.30 ± 0.23 -0.15–0.93
Homocysteine (µmol/L) 13.15 ± 1.97 12.26 ± 1.71 12.72 ± 1.88 9.00–17.00
hsCRP (mg/L) 2.94 ± 3.50 * 1.25 ± 097 2.12 ± 2.70 0.27–15.89
Values are means ± SD; ESR: erythrocyte sedimentation rate, HbA1c : glycated haemoglobin, HOMA-IR: homeostasis
model assessment of insulin resistance, TCHOL: total cholesterol, LDL: low density lipoproteins, AIP: atherogenic
index of plasma, CRP: C-reactive protein; * p < 0.05, significant differences: metabolic syndrome group vs. healthy
(t-test or Mann–Whitney U test).

The most frequently repeating disorders among the criteria adopted in the diagnosis of metabolic
syndrome among the study group were hyperglycaemia, abdominal obesity and hypertension. Detailed
results according to group are presented in Table 2.
J. Clin. Med. 2020, 9, 2797 5 of 21

Table 2. Diagnostics of metabolic syndrome in accordance with NCEP-ATP III in compared groups.

Glucose DBP
Group WC (cm) TG (mg/dL) HDL (mg/dL) SBP (mmHg)
(mg/dL) (mmHg)
Metabolic syndrome 96.24 ± 9.91 * 130.65 ± 41.40 55.50 ± 12.17 * 102.39 ± 9.81 * 127.63 ± 17.27 82.63 ± 7.88
Healthy 84.22 ± 8.71 111.47 ± 38.47 65.06 ± 14.11 92.93 ± 6.94 120.17 ± 16.78 77.83 ± 7.69
Total 88.81 ± 8.58 121.32 ± 40.63 60.15 ± 13.84 97.79 ± 9.69 124.00 ± 17.22 80.30 ± 8.06
Min-Max 73.60−109.80 63.88−215.25 41.41−91.72 73.60−109.80 90−150 60−90
Number of people fulfilling given criterion for diagnosis of metabolic syndrome NCEP-ATP III
Metabolic syndrome 14 (73.7%) 6 (31.6%) 7 (36.8%) 15 (78.9%) 10 (52.6%) 7 (36.8%)
Healthy 3 (16.7%) 3 (16.7%) 1 (5.6%) 3 (16.7%) 6 (33.3%) 4 (22.2%)
Total 17 (45.9%) 9 (24.3%) 8 (21.6%) 18 (48.6%) 16 (43.2%) 11 (29.7%)
Number of metabolic syndrome criteria NCEP-ATP III fulfilled by volunteers
Metabolic
Healthy
syndrome
Number of criteria None One Two Three Four Five
Number of people 4 (10.8%) 10 (27.0%) 4 (10.8%) 11 (29.7%) 4 (10.8%) 4 (10.8%)
Values are means ± SD; NCEP-ATP III: National Cholesterol Education Program-Adult Treatment Panel III, WC:
waist circumference, TG: triglycerides, HDL: high density lipoproteins, SBP: systolic blood pressure, DBP: diastolic
blood pressure; * p < 0.05, significant differences: metabolic syndrome group vs. healthy (t-test or Mann–Whitney
U test).

2.3. Whole-Body Cryotherapy

The examined women underwent 20 WBC sessions, which were performed daily in 4 series of 5
procedures, with a 2-day interval between the series [27]. Treatments were performed at a professional
medical cryotherapy centre in a liquid nitrogen-cooled cryogenic chamber, consisting of a vestibule
(−60 ◦ C) connected directly to the main chamber (−130 ◦ C).
The chamber was equipped with a system for automatic temperature monitoring and air
dehumidification, as well as with oxygen sensors. During the procedure, continuous contact with the
subjects was ensured through the audiovisual system, as well as eye contact through thermal glass in
the chamber door. In order to ensure safety during the procedure, both in the proper chamber and in the
vestibule, there were alarm buttons and mechanical levers enabling the user to immediately open the
door from inside. Treatments were performed under the supervision of qualified physical therapists.
Before beginning the WBC series, the volunteers were informed in detail about how to prepare for
the procedure and how to behave during its implementation.
During the procedures, the women wore a sleeveless cotton tops and shorts that did not put
pressure on the skin; the clothes were devoid of rigid, including metal, elements. Additionally, they
were equipped with a surgical mask with an additional layer of gauze covering the nose and mouth,
woollen socks protecting the ankle joints and knee pads, gloves, a band or a cap protecting the auricles,
and clogs with a wooden sole. The women did not apply cosmetic products to the skin before entering
the cryochamber and thoroughly dried the skin to remove any sweat, the presence of which could
cause frostbite. Before initiating the WBC procedure, the women took off any jewellery or glasses, and
did not use contact lenses.
Each procedure consisted of a 30-second stay in the vestibule of the chamber, followed by a 3 min
stay in the main chamber. Up to 4 people participated in the procedure at a time. During their stay in
the main chamber, the women walked around slowly, changing their direction every 30-second at the
signal. The women were asked to breathe calmly during the procedure, inhaling through their noses,
and exhaling through their mouths, and not to adjust their clothes or touch exposed parts of the body.
Blood pressure was measured before each treatment. In none of the cases did the measurements
exceed 150/90 mmHg, which was adopted as the borderline value allowing for carrying out the WBC
procedure [45]. In addition, blood pressure measurements were also taken after 1, 10 and 20 WBC
treatments. There was no significant effect of single application or a series of 10 and 20 treatments on
systolic or diastolic blood pressure.
J. Clin. Med. 2020, 9, 2797 6 of 21

2.4. Somaticmeasurements and Body Compositionevaluation

Body height was measured to the nearest 1 mm using a stadiometer (Seca 217, Hamburg, Germany).
Body mass (BM) was measured while standing in underwear (Jawon IOI-353 Body composition
Analyzer, Gyeongsa, Korea).
Circumference was measured in a standing position to the nearest 1 mm, placing an anthropometric
tape (Seca 201, Hamburg, Germany) perpendicular to the vertical axis of the body. Waist circumference
(WC) was measured at the narrowest point between the lower edge of the costal arches and the highest
point of the iliac crests. Abdominal circumference (AC) was measured approximately 1 cm below
the navel. Hip circumference (HC) was measured above the buttocks at the widest point around
the greater trochanter, making sure not to be lower than the pubic symphysis. Waist and abdominal
circumferences were measured at the end of calm exhalation.
Skin fold thickness was measured in a standing position using the calliper method to the nearest
0.1 mm (Harpenden skinfold calliper, Burgess Hill, UK) on the abdomen, at the lower angle of the
scapula and above the triceps muscle of the upper limb.
All measurements of circumferences and skin fold thickness were taken 3 times by the same
person with extensive experience, always using the same anthropometric tape and the same calliper.
In the analysis, the average of the two closest results was taken into account.
Body composition was measured by dual energy X-ray absorptiometry (DXA) (GE Healthcare
Lunar iDXA, Madison, WI, USA) which, according to the manufacturer’s instructions, was calibrated
each day before the series of measurements. During the measurement, the women wore clothes that
did not restrict the waist area (cotton sleeveless top and shorts), and were asked to remove plastic,
rubber, metal objects and jewellery. The examination was performed in supine, immobile position,
with the upper limbs extended along the body, the palms of the hands pointing towards the thighs.
During the examination, segmental assessment of body composition was performed.
For each of the participants, Body Mass Index (BMI), Waist-Hip Ratio (WHR) and Waist-Height
Ratio were calculated according to the formulae:

BMI = body mass (kg)/(body height (m))2 (1)

WHR = waist circumference (cm)/hip circumference (cm) (2)

WHtR = waist circumference (cm)/body height (cm) (3)

2.5. Biochemical Analysis

The concentrations of IL-6 (EDTA) and irisin (EDTA and aprotinin 0.6 TIU/1 mL of blood) were
determined in the plasma, and the concentration of CRP in the serum (clotting activator). Venous blood
was collected using a vacuum system (Becton Dickinson, Franklin Lakes, NJ, USA) and centrifuged
(RCF 1.000× g) for 15 min at 4 ◦ C (MPW-351R, MPW Med. Instruments, Warsaw, Poland). The
supernatant was stored until analysis at −70 ± 5 ◦ C (ZLN-UT 300 PREM, POL-EKO-APARATURA,
Wodzislaw Slaski, Poland).
The concentrations of IL-6 and irisin were determined via enzymatic immunoassay (ELISA),
according to manufacture guidelines, using the highly sensitive HS600B test (R&D Systems, Inc.,
Minneapolis, MN, USA) and Irisin/FNDC5 EK-067-16 (Phoenix Pharmaceuticals, Inc., Burlingame, CA,
USA). The results were read from a standard curve created during each of the determinations. The
sensitivity of the assay for IL-6 was 0.039 pg/mL, while the intra-assay CV < 7.8%, and the inter-assay
CV IL-6 < 7.2%. The detection range for irisin was 0–100 ng/mL. CRP concentration was determined
via the immune turbidimetric method using the Cardiac C-Reactive Protein (Latex) High Sensitive
Test-CRPHS (Roche Diagnostics GmbH, Mannheim, Germany). The detection range of the CRPHS
assay was 0.15–20 mg/L, while for the inter-assay, CV < 10%.
J. Clin. Med. 2020, 9, 2797 7 of 21

2.6. Assessment of Physicalactivity

Before the first application and in the last week of using WBC, the women’s physical activity (PA)
was assessed using the short version of the International Physical Activity Questionnaire (IPAQ)-Polish
version, according to the scale: high, moderate or low. The IPAQ contains 7 questions on PA related to
everyday life, work and leisure, performed within the 7 days preceding the survey [46].
Prior to WBC, it was found that 11 people (58%) in the MetS group exhibited moderate PA, while
8 (42%) demonstrated low PA. In the group of healthy women, 9 participants were characterised
by moderate (50%) physical activity, and for 9, this level was low (50%). The PA did not change
significantly during the period WBC implementation. At the end of the study, moderate PA was
declared by 10 people in the MetS group and 8 healthy people, while low PA was affirmed by 12 and
10 people in the MetS and healthy groups, respectively. None of the women demonstrated high PA.

2.7. Assessment of Nutritional Behaviour

Since the change in caloric content and composition of the diet can significantly affect the change
in body composition [47] and in the level of irisin [48,49], the volunteers were asked not to change
their diet and to record the composition of the meals consumed on a daily basis. The portion size was
determined based on a photo album of products and dishes [50]. Nutrient and energy consumption
were calculated (Dieta 5.0, Institute of Food and Nutrition, Warsaw, Poland). The menus were assessed
in relation to the nutritional standards for the Polish population developed by the Institute of Food and
Nutrition [44]. The daily (24-h) dietary caloric intake before the 1st session WBC was 1911.85 ± 385.48
kcal/d and 1735.26 ± 333.72 kcal/d, respectively in the MetS group and in healthy women. The daily
supply of protein, carbohydrates and fat in the diet before WBC1 in the MetS group was: 16.22 ± 4.14%,
54.31 ± 12.28% and 27.19 ± 9.21%. In the group comprising health women, these values totalled: 17.20
± 4.35%, 54.58 ± 11.17% and 25.68 ± 7.32%, respectively. The menus from the week before beginning
WBC treatments (Pre 1 WBC) and the week before the 10th (Pre 10 WBC) and 20th WBC (Pre 20 WBC)
procedures were assessed. There were no differences in the caloric or main nutrient intake within or
between groups during the study period (p > 0.05).

2.8. Statistical Analysis

The distribution of results for the analysed variables was checked using the Shapiro–Wilk test, and
the equality of variance with the Levene test. For single measurements, the significance of group-related
differences was assessed using independent-sample tests, Student’s t-test or the Mann–Whitney U test.
Pearson or Spearman correlations were calculated.
Comparing the impact of WBC treatments on changes in the analysed variables among the
compared groups, analysis of variance with repeated measures (ANOVA) was used, examining the
influence of the main factors, i.e., Group (MetS and Healthy), Treatment (influence of WBC) and Group
× Treatment interaction. Effect sizes for ANOVA analysis were calculated using partial eta squared
(η2 ) and interpreted as 0.010–0.059 = small, 0.060–0.139 = medium, ≥0.14 = large. For changes in the
level of specific variables after WBC, confidence intervals were determined (95% CI). When significant
influence of the main factors was found, post-hoc analysis was performed using the Fisher LSD test.
The statistical significance of differences was assumed for the level of p < 0.05. The statistical power of
the test was also calculated (1-β). The STATISTICA 13 package (StatSoft, Inc., Tulsa, OK, USA) was
used for calculations.
J. Clin. Med. 2020, 9, 2797 8 of 21

3. Results

3.1. Changes in Body Composition, Circumferences and Skin Fold Thicknesses as an Effect of Applying
Whole-Body Cryotherapy Procedures

3.1.1. Body Mass and Composition

A significant effect of WBC was noted (ANOVA Treatment, large effect size) on changes in BM
(η2 = 0.17, p = 0.001), BMI (η2 = 0.17, p = 0.001) and total body fat mass (η2 = 0.19, p = 0.001), as well as
in the percentage value of fat in the lower limbs (η2 = 0.14, p = 0.006), while a medium effect size was
observed for changes in percentage value of fat content in total body mass (η2 = 0.09, p = 0.036) as well
as in the abdominal (android) area (η2 = 0.08, p = 0.049) and trunk (η2 = 0.06, p = 0.113) (Table 3).
After 20 WBC treatments, in the MetS group and in the group of healthy women, significant
reductions were noted (post-hoc, p < 0.05) in BM (−0.56: 95% CI −1.20; 0.08 and −0.67: 95% CI −0.98;
−0.35, respectively), BMI (−0.24: 95% CI −0.49; 0.02 and −0.25: 95% CI −0.37; 0.14, respectively) and
total fat mass (−0.50: 95% CI −0.93; −0.07 and −0.51: 95% CI −0.89; −0.12, respectively), as well as
in the percentage of fat in the lower limbs (−0.55: 95% CI −1.01; −0.09 and −0.51: 95% CI −1.10; 0.09,
respectively). Additionally, in the MetS group, after 20 WBC sessions, the percentage of adipose tissue
in the total body mass decreased (−0.48: 95% CI −0.91; −0.05) significantly (post-hoc, p < 0.05) as well
as in the trunk (−0.64: 95% CI −1.34; 0.06) and android area (−1.08: 95% CI −1.98; −0.19) (Table 4).
Significant differences between groups were noted (ANOVA Group, large effect size) in the level
of BM (η2 = 0.25, p = 0.001), BMI (η2 = 0.27, p = 0.001), LBM (η2 = 0.16, p = 0.014), total body fat mass
(η2 = 0.31, p < 0.001), percentage of fat in total body mass (η2 = 0.21, p = 0.005) and the percentage of
fat in the trunk (η2 = 0.23, p = 0.003) as well as abdominal areas (η2 = 0.21, p = 0.004), and furthermore,
in the percentage of fat in the android/gynoid sections (η2 = 0.15, p = 0.020) (Table 3).
Both before the 1st WBC application as well as after 10 and after 20 WBC treatments, the level of
these variables was significantly higher (post-hoc, p < 0.05) in the MetS group compared to the group
of healthy women (Table 4).
No influence of Group×Treatment factor interaction on the level of indices was demonstrated
(Tables 3 and 4).

3.1.2. Circumferences
Significant influence of WBC (ANOVA Treatment, large effect size) on changes in WC (η2 = 0.33, p
< 0.001), AC (η2 = 0.22, p = 0.001), HC (η2 = 0.26, p < 0.001) and WHtR (η2 = 0.33, p < 0.001) was noted
(Table 3).
In both groups, there was a significant decrease (post-hoc, p < 0.05) in WC and WHtR after 10
WBC (−2.17: 95% CI −3.78; −0.56 and −0.01: 95% CI −0.02; −0.00 as well as −1.28: 95% CI −2.30; −0.25
and −0.01: 95% CI −0.01; 0.00, respectively, in the MetS and healthy groups), and 20 WBC treatments
(−1.96: 95% CI −3.38; −0.55 and −0.01: 95% CI −0.02; −0.00 as well as −1.57: 95% CI −2.63; −0.50 and
−0.01: 95% CI −0.02; 0.00, respectively, in the MetS and healthy groups), and a significant decrease
(post-hoc, p < 0.05) in AC only after 20 WBC sessions (−2.47: 95% CI −5.25; −0.30 and −2.17: 95% CI
−3.69; −0.66, respectively, in the MetS and healthy groups). A significant reduction (post-hoc, p < 0.05)
in HC was also found in the group of healthy women after 10 WBC (−1.04: 95% CI −1.86; −0.22) and
20 WBC sessions (−1.79: 95% CI −2.54; −1.05) (Table 4).
Significant differences between groups were noted (ANOVA Group, large effect size) in the level
of WC (η2 = 0.31, p = 0.002), AC (η2 = 0.27, p = 0.004), HC (η2 = 0.27, p = 0.004) and WHtR (η2 = 0.30, p
= 0.002) (Table 3).
In all measurements, WC, AC, HC and WHtR were significantly higher (post-hoc, p < 0.05) in the
MetS group, compared to the group comprising healthy women (Table 4).
No influence of Group × Treatment factor interaction on the level of analysed indices was
demonstrated (Tables 3 and 4).
J. Clin. Med. 2020, 9, 2797 9 of 21

Table 3. Assessment regarding the effects of whole-body cryotherapy (WBC) treatments on changes in body composition markers-analysis of variance with repeated
measures (ANOVA).

Group (G) Treatment (T) Interaction G×T

Variable
Power Effect Power Effect Power Effect
p F p F p F
Test 1-β Size η2 Test 1-β Size η2 Test 1-β Size η2
Body composition
Body Mass (kg) 0.001 11.98 0.92 0.25 0.001 7.31 0.93 0.17 0.780 0.25 0.09 0.01
BMI (kg/m2 ) 0.001 12.62 0.93 0.27 0.001 7.47 0.93 0.18 0.824 0.19 0.08 0.01
Lean Body Mass (kg) 0.014 6.74 0.71 0.16 0.760 0.28 0.09 0.01 0.623 0.48 0.12 0.01
Total Body Fat (kg) <0.001 15.49 0.97 0.31 0.001 8.42 0.96 0.19 0.954 0.05 0.06 <0.01
Total Body Fat (%) 0.005 9.04 0.83 0.21 0.036 3.50 0.63 0.09 0.840 0.18 0.08 <0.01
Arm Fat (%) 0.216 1.59 0.23 0.04 0.221 1.54 0.32 0.04 0.976 0.02 0.05 <0.01
Leg Fat (%) 0.091 3.01 0.39 0.08 0.006 5.59 0.84 0.14 0.986 0.01 0.05 <0.01
Trunk Fat (%) 0.003 10.32 0.88 0.23 0.113 2.25 0.44 0.06 0.654 0.43 0.12 0.01
Android Fat (%) 0.004 9.52 0.85 0.21 0.049 3.06 0.57 0.08 0.484 0.73 0.17 0.02
Gynoid Fat (%) 0.098 2.88 0.38 0.08 0.277 1.31 0.27 0.04 0.353 1.06 0.23 0.03
A/G 0.020 5.94 0.66 0.15 0.314 1.18 0.25 0.03 0.792 0.23 0.09 0.01
Circumferences
WC (cm) 0.002 11.94 0.91 0.31 <0.001 13.54 0.99 0.33 0.516 0.67 0.16 0.02
AC (cm) 0.004 9.95 0.86 0.27 0.001 7.52 0.93 0.22 0.471 0.76 0.17 0.03
HC (cm) 0.004 9.93 0.86 0.27 <0.001 9.67 0.98 0.26 0.372 1.01 0.22 0.04
WHR 0.124 2.52 0.35 0.09 0.080 2.71 0.51 0.09 0.363 1.03 0.22 0.04
WHtR 0.002 11.60 0.91 0.30 <0.001 13.19 0.99 0.33 0.521 0.66 0.16 0.02
Skinfold thickness
Abdominal (mm) 0.030 5.12 0.59 0,13 0.002 6.94 0.91 0.17 0.052 3.09 0.58 0.08
Subscapular (mm) 0.214 1.60 0.23 0.04 0.643 0.45 0.12 0.01 0.572 0.56 0.14 0.02
Triceps (mm) 0.003 9.96 0.87 0.22 <0.001 9.84 0.98 0.22 0.357 1.04 0.23 0.03
p < 0.05: statistically significant differences; BMI: body mass index, A/G: android-to-gynoid percentage fat ratio; WC: waist circumference, AC: abdominal circumference, HC: hip
circumference, WHR: waist-hip ratio; WHtR: waist–height ratio.
J. Clin. Med. 2020, 9, 2797 10 of 21

Table 4. Changes in body composition, circumferences and fatfold thickness as an effect of applying whole-body cryotherapy (WBC) treatments.

Metabolic Syndrome Healthy

Pre After After Pre After After
Variable ∆ 10 WBC ∆ 20 WBC ∆ 10 WBC ∆ 20 WBC
1 WBC 10 WBC 20 WBC 1 WBC 10 WBC 20 WBC
Mean ± SD Mean ± SD Mean ± SD Mean (95% CI) Mean (95% CI) Mean ± SD Mean ± SD Mean ± SD Mean (95% CI) Mean (95% CI)
Body composition
Body Mass (kg) 77.36 ± 11.95 76.97 ± 12.06 76.80 ± 12.13 * −0.38 (−0.89; 0.12) −0.56 (−1.20; 0.08) 66.32 ± 6.23 ‡ 66.06 ± 6.50 ‡ 65.66 ± 6.32 *,‡ −0.27 (−0.64; 0.11) −0.67 (−0.98; −0.35)
BMI (kg/m2 ) 30.09 ± 4.98 29.93 ± 4.93 29.86 ± 4.94 * −0.16 (−0.36; 0.03) −0.24 (−0.49; 0.02) 25.50 ± 2.37 ‡ 25.39 ± 2.45 ‡ 25.24 ± 2.43 *,‡ −0.11 (−0.25; 0.04) −0.25 (−0.37; 0.14)
Lean Body Mass (kg) 44.19 ± 4.75 44.14 ± 5.11 44.21 ± 4.83 −0.47 (−0.59; 0.50) 0.24 (−0.43; 0.48) 40.76 ± 3.31 ‡ 40.68 ± 3.28 ‡ 40.52 ± 2.96 ‡ −0.08 (−0.43; 0.27) −0.24 (−0.65; 0.17)
Total Body Fat (kg) 34.42 ± 8.58 34.15 ± 8.55 33.92 ± 8.81 * −0.27 (−0.61; 0.08) −0.50 (−0.93; −0.07) 25.52 ± 4.08 ‡ 25.32 ± 4.33 ‡ 25.01 ± 4.33 *,‡ −0.20 (−0.53; 0.12) −0.51 (−0.89; −0.12)
Total Body Fat (%) 42.42 ± 4.32 42.21 ± 4.45 41.93 ± 4.59 * −0.21 (−0.69; 0.28) −0.48 (−0.91; −0.05) 38.38 ± 3.48 ‡ 38.22 ± 3.63 ‡ 38.07 ± 3.54 ‡ −0.16 (−0.56; 0.25) −0.31 (−0.84; 0.22)
Arm Fat (%) 42.67 ± 4.37 43.18 ± 3.96 42.87 ± 4.18 0.51 (−0.42; 1.44) 0.20 (−0.52; 0.92) 41.06 ± 3.88 41.51 ± 4.44 41.14 ± 4.02 0.44 (−0.55; 1.44) 0.08 (−0.73; 0.89)
Leg Fat (%) 39.62 ± 4.99 39.40 ± 5.08 39.07 ± 5.01 * −0.22 (0.73; 0.29) −0.55 (−1.01; −0.09) 36.99 ± 3.97 36.82 ± 4.13 36.49 ± 4.07 * −0.17 (−0.52; 0.18) −0.51 (−1.10; 0.09)
Trunk Fat (%) 46.54 ± 5.08 46.21 ± 5.42 45.90 ± 5.69 * −0.33 (−1.01; 0.34) −0.64 (−1.34; 0.06) 41.02 ± 4.88 ‡ 40.72 ± 4.85 ‡ 40.75 ± 4.89 ‡ −0.31 (−0.91; 0.30) −0.27 (−0.98; 0.43)
Android Fat (%) 48.98 ± 5.93 48.40 ± 6.34 47.90 ± 6.57 * −0.58 (−1.39; 0.22) −1.08 (−1.98; −0.19) 42.74 ± 5.58 ‡ 42.25 ± 5.82 ‡ 42.34 ± 5.61 ‡ −0.49 (−1.36; 0.38) −0.39 (−1.45; 0.66)
Gynoid Fat (%) 44.24 ± 4.66 43.90 ± 4.67 43.69 ± 4.90 −0.34 (−0.91; 0.23) −0.55 (−1.14; 0.05) 41.0 ± 3.40 41.69 ± 3.58 41.55 ± 3.74 0.09 (−0.47; 0.66) −0.05 (−0.68; 0.58)
A/G 1.11 ± 0.10 1.10 ± 0.09 1.10 ± 0.11 −0.01 (−0.03; 0.01) −0.01 (−0.04; 0.02) 1.03 ± 0.11‡ 1.01 ± 0.12 ‡ 1.02 ± 0.11 ‡ −0.01 (−0.03; 0.00) −0.01 (−0.03; 0.01)
Circumferences
WC (cm) 96.24 ± 9.91 94.06 ± 8.25 * 94.27 ± 9.96 * −2.17 (−3.78; −0.56) −1.96 (−3.38; −0.55) 84.22 ± 8.71 ‡ 82.94 ± 8.46 *,‡ 82.66 ± 8.27 *,‡ −1.28 (−2.30; −0.25) −1.57 (−2.63; −0.50)
AC (cm) 104.94 ± 10.98 103.16 ± 9.76 102.46 ± 10.76 * −1.77 (−4.82; 1.27) −2.47 (−5.25; −0.30) 93.91 ± 7.40 ‡ 93.54 ± 7.6 ‡ 91.73 ± 7.77 *,‡ −0.37 (−1.66; 0.93) −2.17 (−3.69; −0.66)
101.09 ± 5.43
HC (cm) 112.06 ± 11.71 111.21 ± 11.90 111.13 ± 12.24 −0.85 (−2.07; 0.36) −0.94 (−2.30; 0.42) 102.13 ± 6.18 ‡ 100.34 ± 5.4 *,‡ −1.04 (−1.86; −0.22) −1.79 (−2.54; −1.05)
*,‡
WHR 0.86 ± 0.04 0.85 ± 0.03 0.85 ± 0.04 −0.01 (−0.02; 0.00) −0.01 (−0.02; −0.00) 0.82 ± 0.06 0.82 ± 0.06 0.82 ± 0.06 0.00 (−0.01; 0.00) 0.00 (−0.01; 0.00)
WHtR 0.59 ± 0.06 0.58 ± 0.05* 0.58 ± 0.06* −0.01 (−0.02; −0.00) −0.01 (−0.02; −0.00) 0.52 ± 0.05 ‡ 0.51 ± 0.05 *,‡ 0.51 ± 0.05 *,‡ −0.01 (−0.01; 0.00) −0.01 (−0.02; 0.00)
Skinfold thickness
Abdominal (mm) 29.44 ± 8.65 29.66 ± 8.26 28.56 ± 8.17 0.22 (−0.63; 1.06) −0.88 (−1.85; 0.08) 25.17 ± 7.12 ‡ 23.18 ± 7.08 *,‡ 22.61 ± 6.23 *,‡ −1.98 (−4.05; 0.08) −2.56 (−4.75; −0.36)
Subscapular (mm) 24.91 ± 7.95 24.91 ± 7.48 24.96 ± 7.48 0.01 (−0.43; 0.44) 0.06 (−0.66; 0.78) 21.87 ± 6.76 22.24 ± 6.20 21.88 ± 6.38 0.37 (−0.36; 1.11) 0.02 (−0.79; 0.83)
Triceps (mm) 24.31 ± 4.95 24.30 ± 4.73 23.77 ± 4.63 * −0.01 (−0.45; 0.44) −0.54 (−1.09; 0.01) 19.84 ± 4.90‡ 19.41 ± 4.47 ‡ 18.83 ± 4.05 *,‡ −0.43 (−1.03; 0.17) −1.01 (−1.82; −0.20)
SD: standard deviation, CI: confidence interval, ∆ 10 WBC: difference after 10 WBC compared to pre 1 WBC, ∆ 20 WBC: difference after 20 WBC compared to pre 1 WBC; BMI: body mass
index, A/G: android-to-gynoid percentage fat ratio; WC: waist circumference, AC: abdominal circumference, HC: hip circumference, WHR: waist-hip ratio; WHtR: waist-height ratio; *
statistically significant differences compared to values pre 1 WBC (p < 0.05); ‡ statistically significant differences between groups (p < 0.05).
J. Clin. Med. 2020, 9, 2797 11 of 21

3.1.3. Skinfolds
Significant influence of WBC was noted (ANOVA Treatment, large effect size) on changes in
abdominal thickness (η2 = 0.17, p = 0.002) and triceps skinfolds (η2 = 0.22, p < 0.001) (Table 3).
The thickness of the triceps skinfold in both groups decreased significantly (post-hoc, p < 0.05)
after 20 WBC procedures (−0.54: 95% CI −1.09; 0.01 and −1.01: 95% CI −1.82; −0.20, respectively, in the
MetS and healthy groups). In the group of healthy women, the thickness of the abdominal skinfold
was significantly (post-hoc, p < 0.05) lower after 10 WBC (−1.98: 95% CI −4.05; 0.08) and 20 WBC
treatments (−2.56: 95% CI −4.75; −0.36) (Table 4).
Significant differences between groups were noted (ANOVA Group) in abdominal thickness (η2 =
0.13, p < 0.030) and triceps skinfolds (η2 = 0.22, p = 0.003) (Table 3).
For all measurements, the thickness of abdominal and triceps skinfolds was significantly greater
(post-hoc, p < 0.05) in the MetS group compared to healthy women (Table 4).
No influence of Group × Treatment factor interaction on the level of analysed indices was
demonstrated (Tables 3 and 4).

3.2. Irisin, IL-6 and CRP Concentrations during the Application of Whole-Body Cryotherapy
In Figure 1, a comparison of irisin, IL-6 and CRP concentrations in the metabolic syndrome group
and in the group of healthy women during whole-body cryotherapy implementation is shown.

3.2.1. Irisin
Irisin concentration in the plasma of women with MetS was 1.87 ± 0.24 µg/mL, 2.05 ± 0.25 µg/mL,
2.03 ± 0.22 µg/mL and 1.92 ± 0.19 µg/mL before and after the 1st WBC session, as well as after the 10th
and 20th WBC treatments. In the group of healthy women, the respective values were 1.89 ± 0.22
µg/mL, 2.10 ± 0.12 µg/mL, 2.03 ± 0.26 µg/mL and 1.96 ± 0.21 µg/mL. There were no differences within
the groups related to irisin concentration (ANOVA Group; p = 0.546, F = 0.37, η2 = 0.01, 1-β = 0.09). A
significant effect of WBC on irisin concentration was found (ANOVA Treatment; p < 0.001, F = 6.76, η2
= 0.16, 1-β = 0.97). There was no significant interaction of Group×Treatment factors (ANOVA, p =
0.948, F = 0.12, η2 < 0.01, 1-β = 0.07).
In the group of women with MetS, the concentration of irisin after 1 WBC (post-hoc, p = 0.007) and
10 WBC sessions (post-hoc, p = 0.017) was significantly higher than the baseline value. The differences
totalled 0.18 (95% CI 0.03; 0.32) and 0.16 (95% CI 0.02; 0.34), respectively. Similarly, in the group of
healthy women, after 1 WBC (post-hoc, p = 0.003) and 10 WBC sessions (post-hoc, p = 0.049), a higher
concentration of irisin was detected compared to baseline (respectively, by 0.20: 95% CI 0.09; 0.31
and 0.13: 95% CI 0.01; 0.28). The concentration of irisin after 20 WBC treatments was comparable to
baseline (post-hoc, p > 0.05), the differences totalling 0.04 (95% CI −0.8; 0.17) and 0.07 (95% CI −0.03;
0.16) in the MetS and health-women groups, respectively.
J. Clin. Med. 2020,
J. Clin. 9, 2797
Med. 2020, 9, x FOR PEER REVIEW 13 of 22 12 of 21

2.15 7 4.5 Pre 1 WBC After 1 WBC After 10 WBC After 20 WBC
Pre 1 WBC After 1 WBC After 10 WBC After 20 WBC Pre 1 WBC After 1 WBC After 10 WBC After 20 WBC
‡
4.0 p=0.018
2.10 * p=0.007 ‡
6 p=0.033
* p=0.003 ‡
p=0.038
* p<0.001 3.5
2.05 ‡

C-reactive protein (mg/L)

* p=0.049 * p=0.017 p=0.002
5

Interleukin-6 (pg/mL)
3.0
* p=0.001
Irisin (µg/mL)

2.00 ‡
p=0.012
4 2.5

1.95
2.0
3
1.90
1.5

1.85 2
1.0

1.80 1 0.5
Metabolic syndrome Healthy Metabolic syndrome Healthy Metabolic syndrome Healthy

(a) (b) (c)

Figure 1.Figure 1. Comparison

Comparison of concentrations:
of concentrations: (a) Plasma
(a) Plasma irisin;
irisin; (b)plasma
(b) plasmainterleukin-6;
interleukin-6; (c)
(c)serum
serumC-reactive protein
C-reactive in the
protein in group with metabolic
the group syndrome
with metabolic and in the
syndrome and in the
group of healthy women during the application of whole-body cryotherapy (WBC) procedures. Data presented as mean ± standard deviation;
group of healthy women during the application of whole-body cryotherapy (WBC) procedures. Data presented as mean ± standard deviation; * statistically significant * statistically
significant
differences compareddifferences compared
to values to1st
prior to values
WBC prior to 1st (p
session WBC< 0.05);
session (p < 0.05); ‡ statistically
‡ statistically significantsignificant differences
differences betweenbetween (p < 0.05).
groupsgroups (p < 0.05).

J. Clin. Med.2020, 9, x; doi: FOR PEER REVIEW www.mdpi.com/journal/jcm

J. Clin. Med. 2020, 9, 2797 13 of 21

3.2.2. Interleukin-6
The concentration of IL-6 in the plasma of women with MetSwas 1.78 ± 0.84 pg/mL, 1.58 ± 0.72
pg/mL, 4.00 ± 4.11 pg/mL and 4.77 ± 4.35 pg/mL before WBC 1, after WBC 1, after WBC 10 and after
WBC 20, respectively. In the group of healthy women, the respective values were 2.06 ± 1.17 pg/mL,
1.70 ± 0.94 pg/mL, 2.08 ± 0.68 pg/mL and 2.40 ± 1.14 pg/mL. There was a significant group effect
(ANOVA Group; p = 0.045, F = 4.33, η2 = 0.11, 1-β = 0.52), WBC (ANOVA Treatment; p < 0.001, F =
7.20, η2 = 0.17, 1-β = 0.98) and the interaction of the Group×Treatment factors (ANOVA, p = 0.009, F =
4.03, η2 = 0.10, 1-β = 0.83) on the concentration of IL-6 in the plasma.
The IL-6 concentration increased significantly after the 10 WBC (post-hoc, p = 0.001) and after
the 20 WBC procedures (post-hoc, p < 0.001), but only in women with MetS (2.22: 95% CI 0.15; 4.29
and 2.99: 95% CI 0.73; 5.26, respectively). The change in IL-6 concentration after 1 WBC application in
the MetS group was not statistically significant (post-hoc, p > 0.05) and totalled −0.19 (95% CI −0.38;
−0.01). In the group of health women, changes in IL-6 concentrations were non-significant (post-hoc, p
> 0.05) and totalled −0.36 (95% CI −0.68; −0.05), 0.01 (95% CI −0.66; 0.69) and 0.33 (95% CI −0.32; 0.99),
after 1, 10 and 20 WBC treatments, respectively.
Baseline plasma levels of IL-6 were similar in both groups (p > 0.05), while after WBC 10 (p =
0.012) and WBC 20 (p = 0.002), they were higher in women with MetS compared to the healthy subjects.

3.2.3. C-Reactive Protein

The CRP concentration in the serum of women with MetS was 2.94 ± 3.50 mg/L, 2.85 ± 2.90 mg/L,
3.04 ± 3.86 mg/L and 2.39 ± 1.72 mg/L (respectively) before and after WBC 1, after WBC 10 and after
WBC 20. In the group of healthy women, the respective values were 1.25 ± 0.97 mg/L, 1.20 ± 0.85
mg/L, 1.16 ± 0.99 mg/L and 1.39 ± 1.44 mg/L. There were significant group-related differences in CRP
concentration (ANOVA Group; p = 0.030, F = 5.13, η2 = 0.13, 1-β = 0.60), however, no effects of WBC
(ANOVA Treatment; p = 0.880, F = 0.22, η2 = 0.01, 1-β = 0.09) or Group×Treatment (ANOVA p = 0.460,
F = 0.87, η2 = 0.02, 1-β = 0.23) were noted on the CRP concentration in these groups.
The CRP concentration in the serum before (post-hoc, p = 0.033), after the 1 WBC (post-hoc, p =
0.038) and after the 10 WBC session (post-hoc, p = 0.018) was significantly higher in the MetS group
compared to the healthy women. After 20 WBC treatments, the CRP concentration was comparable in
both groups (p > 0.05).

3.3. Correlations
In the MetS group and in the group of healthy women, the correlation between the concentration
of irisin, IL-6, and between changes in their concentration and statistically significant changes in
somatic constitution indices was assessed.
In the MetS group, a significant negative correlation was found between the change in irisin
concentration after 10 WBC treatments, and between the change in HC (r = −0.84, p < 0.05). Furthermore,
between the change in irisin concentration after 20 WBC sessions and the change in body mass (r =
−0.66, p < 0.05), as well as between the concentration of irisin after 20 WBC applications and the change
in total fat percentage (r = −0.48, p < 0.05). There was also a significant negative correlation between
the change in IL-6 concentration after 10 WBC procedures and a change in WHR (r = −0.65, p < 0.05),
WHtR (r = −0.64, p < 0.05) and WC (r = −0.63, p < 0.05). No such correlations were found in the group
of healthy women.

4. Discussion
A factor strongly affecting the human body is cryogenic temperature used during WBC [30]. Our
study was the first to show that WBC can effectively support the treatment of abdominal obesity in
menopausal women. It has been proven that the first beneficial changes occur after 10 WBC treatments
and apply to both menopausal women with MetS and without severe metabolic disorders. Increasing
J. Clin. Med. 2020, 9, 2797 14 of 21

the number of WBC treatments to 20 increased the beneficial changes in body composition among
menopausal women.
In our study, 20 WBC treatments were used, which were applied daily in 4 series of 5 treatments,
between which there were 2-day intervals (weekends). The procedures were performed among
menopausal women in whom MetS occurs more often than in young women and men [6,16]. On the
other hand, previous research on the influence of WBC on the body of obese individuals, conducted by
other scientists, concerned men [24–26,28]. In order to eliminate additional factors that may affect the
obtained results [51], during the period of using WBC, the women in our study did not change their
diets or physical activity, which they declared to be at a low or sufficient level [46].
The basic method of assessing overweightness and obesity is BMI [1]. In our study, in both groups,
after 20 WBC treatments, the body mass of the participants decreased and, consequently, the BMI
value also experienced a significant reduction. After the procedures, the BMI level in 26 subjects
was lower than before cryotherapy. But only in one of the participants undergoing therapy did BMI
normalisation occur. However, the knowledge of BMI values does not allow determination of adipose
tissue distribution.
Although according to current criteria, abdominal obesity is not a necessary condition for the
diagnosis of MetS [10–13], besides arterial hypertension, it is the most common disorder in this
disease [19]. Abdominal obesity leads to life-threatening consequences. It influences the development
of insulin resistance and disorders of lipid metabolism, leading to excessive oxidation of fatty acids
and consequently, to oxidative stress and the formation of atherosclerotic plaques, the development of
hypertension and chronic inflammation [9,18,19,52].
Visceral fat content can be estimated by measuring waist circumference and determining the
waist-to-hip ratio as well as the waist-to-body height ratio [53,54]. In our study, a waist circumference
above 88 cm, which was one of the MetS diagnostic criteria according to NCEP - ATP III, was present
in as many as 45.9% of all participants. Among women with MetS, as many as 73.7% met this criterion.
It was found that after 10 WBC treatments there was a reduction in WC and WHtR in both of the
compared groups, and in healthy menopausal women the hip circumference and abdominal skinfold
thickness were also reduced. After 20 WBC procedures, in both groups, these favourable changes
intensified. In addition, after 20 WBC units, in both groups, the abdominal circumference and the
thickness of the skin fold above the triceps muscle decreased compared to the value measured before
cryotherapy. Based on the values of confidence intervals, it may be concluded that in these cases, the
direction of changes was uniform in each group. A large effect size regarding the changes was also
found for these variables, the greatest in WC and WHtR. The results obtained prove the beneficial
influence of WBC in the battle against obesity. Also, in young obese men, the beneficial effect of 20 WBC
treatments can been seen in the reduction of WC and HC [28]. However, in these studies, the influence
of diet and physical activity on the results cannot be ignored. Researchers advised participants not to
change their habits, however, this was not controlled during the application of WBC [28]. In our study,
diet and physical activity were monitored during the use of WBC, and it was found that they were not
significantly modified in any of the groups. This excludes the possibility of these factors affecting our
obtained results.
In our study we assessed the influence of WBC on body composition and adipose tissue distribution
using the dual DXA. It was found that in both groups including menopausal women, weight loss after
the 20th WBC session was the result of a significant reduction in body fat mass. Analysing the changes
in the distribution of adipose tissue in individual body segments, it was noted that in both groups,
the percentage of adipose tissue in the lower limbs decreased. In the group of women with MetS, the
beneficial effects of cryotherapy also consisted in a significant reduction in the percentage of body fat
in the entire trunk area, but also in the percentage of abdominal fat. The analysis of results indicates
a one-way, clinically beneficial (large or medium effect size) changes concerning the majority of the
analysed variables.
J. Clin. Med. 2020, 9, 2797 15 of 21

The number of exposures to cryogenic temperatures seems to be significant in influencing changes

regarding body composition. As in the case of women in our study, the use of 10 WBCs in young men
did not affect the total body mass or the percentage of body fat [55]. In this study [55], WBC treatments
lasting 2 min at −110 ◦ C were performed twice a day for 5 consecutive days. Changes in body
composition after the application of 10 WBC treatments performed daily for 3 min at a temperature of
−110◦ C in obese men around the age of 40, were also not found by Ziemann et al. [25], who compared
the results of people with lower (VO2max 25.7 ± 2.0 mL/kg/min, BMI 34.0 ± 2.5 kg/m2 ) and higher
cardiorespiratory fitness (VO2max 43.3 ± 3.0 mL/kg/min, BMI 31.4. ± 2.0 kg/m2 ). Also, Lubkowska
et al. [24] did not find any significant changes in body composition as a result of WBC application,
although the treatments were supported by physical training. In these studies, the volunteers were
obese men (40 ± 4 years, BMI 30.39 ± 4.31 kg/m2 ) who participated in a 6-month training cycle
with moderate intensity exercises 3 times a week. In the 2nd and 6th month of training, they were
additionally subjected to 20 WBC treatments (3 min, −130 ◦ C). Waist and hip circumferences and
WHR did not change significantly. There were also no changes to body fat mass, subcutaneous fat
mass, visceral fat mass, LBM, skeletal muscle mass, or the percentage of these components during the
study [24]. However, in research involving younger men with low physical activity, both obese (29.08
± 4.19 y, BMI 36.23 ± 8.13 kg/m2 ) and with a normative body mass (22.00 ± 2.45 y, BMI 23.58 ± 2.00
kg/m2 ), as a result of 20 WBC exposures (2–3 min, −120 ◦ C), significant decreases were noted in BM,
fat mass and percentage. In the group of obese men, this resulted in a decrease in BMI [28].
The results of our research indicate the need for at least 20 WBC treatments in a series to achieve
beneficial changes in body composition among menopausal women. In our study, we found that in
both groups of women, WBC treatments had a similar effect, which is indicated by the lack of statistical
significance in the assessment of the interaction of the Group x Treatment factors, with significant
impact of the Treatment factor on the level of the analysed variables. Such results, i.e., the beneficial
effects of WBC on body composition in both groups, are not surprising when considering the results of
previous studies with the participation of men, in which positive changes in body composition of the
participants were also obtained in two groups significantly different in the level of fat and BMI before
beginning the procedures [28]. However, we expected greater clinical effects, especially in the group
of women with MetS. Increasing the number of WBC treatments in a series is likely to intensify the
clinical effect. However, this requires further investigation.
In our study, beneficial changes in body composition in the group of women with MetS were
associated with increases in blood irisin levels. We found that in both groups, the level of irisin
increased significantly after WBC 1 and WBC 10 (large effect size).
The primary physiological mechanism, preventing heat loss in response to cryogenic temperatures,
is the stimulation of the sympathetic nervous system and vasoconstriction in the dermis and
subcutaneous tissue, as well as may induce shivering thermogenesis. During the second phase,
following the procedure, a vasodilation and an increase in perfusion take place [29]. Muscle cell
contractions induce the expression of the FNDC5 gene by increasing PGC1-α levels, which affects the
synthesis of the FNDC5 protein and the release of irisin into the bloodstream. Irisin is a thermogenic
adipomyokine [39]. It has been shown that irisin increases the level of the UCP-1 uncoupling
protein, a slight increase in its concentration causes browning of white adipose tissue, which affects
the thermogenesis process [36]. UCP-1 mediates proton transport across the inner mitochondrial
membrane bypassing ATP synthase. Therefore, energy is dissipated in the form of heat [56]. The
release of heat energy leads to the rapid depletion of cellular storage substrates, which is associated
with weight loss. It has been shown that exogenous irisin stimulates the activation of lipoprotein lipase
and triglyceride lipolysis, enhances lipid metabolism and glucose uptake by cells, and lowers lipid
synthesis in mice [57,58].
Cross-correlations showed that greater increases in irisin levels in our study resulted in greater
weight loss, and higher levels of this hormone were associated with greater reductions in body
fat percentage among women with MetS as a result of cryotherapy. Thus, it has been shown that
J. Clin. Med. 2020, 9, 2797 16 of 21

favourable changes in body composition may be associated with the induction of changes in irisin
concentration by cryogenic temperatures. However, it is surprising that there were no changes in the
concentration of irisin after 20 WBC treatments, which has already been reported by us [27]. A possible
reason for such a reaction is the adaptation to cold and the weakening of shivering thermogenesis
as a probable factor inducing an increase in irisin concentration, which was reported as subjective
sensations by participants.
Dulian et al. [26], who used WBC procedures (3 min, −110 ◦ C) in obese men (BMI > 30 kg/m2 ) aged
38 ± 9 years, found that the change in irisin concentration in response to WBC depends on the level of
physical fitness. Based on the maximum oxygen uptake, the subjects were divided into HFL—high
fitness level (VO2max > 35 mL/kg/min) and LFL—low fitness level (VO2max < 35 mL/kg/min) groups.
After 10 WBC sessions, a slight decrease in irisin concentration was found in the HFL group. However,
in the LFL group, 10 WBC treatments have been found to increase the concentration of irisin in the blood
by about 18% [26]. In the research by Sliwicka et al. [43], a significant increase in the concentration of
irisin in the blood was found in young men with LFL (VO2max < 43 mL/kg/min) in response to the 10th
WBC procedure. In young men, the irisin concentration did not change significantly after the 1st WBC
treatment or after the 10th WBC series [43]. In our study, women from both groups were characterised
by low or moderate physical activity, none of the women practiced high-level physical activity.
In the research by Dulian et al. [26], a positive correlation was indicated between the concentration
of irisin and the mass and percentage of fat as well as with the visceral fat area, and a negative
correlation with the mass of the skeletal muscles. This gave rise to the claim that the main source of
irisin in response to cryogenic exposure is subcutaneous adipose tissue [26], although the results of our
research do not support this conclusion. In this study, no correlation was demonstrated between irisin
levels and adipose tissue or LBM.
The reaction to obesity-related inflammation is the activation of the NF-kB transcription factor,
which enhances, among others, the production of pro-inflammatory interleukins such as IL-1, IL-6 and
TNF-α, and the concentration of C-reactive protein also increases [18,19]. In our study, women with
MetS had significantly higher CRP levels compared to healthy menopausal women, with comparable
IL-6 and irisin levels. In our study, the group of healthy women comprised participants who did not
meet the MetS criteria according to the NCEP-ATP III concept. However, this did not exclude the
occurrence of metabolic disorders (less than 3 criteria), which is the probable cause of similar IL-6 and
irisin concentration levels in both groups.
WBC treatments did not significantly change the CRP concentration in any of the groups with
menopausal women. In contrast, the concentration of IL-6 increased significantly in the MetS group
after 10 and 20 WBC applications (large effect size). The anti-inflammatory effect of whole-body
cryotherapy is demonstrated by an increase in the concentration of irisin and IL-6 without changes
in the concentration of CRP in the blood. As in this study, there were no significant changes in CRP
levels after 10 WBC treatments in 20-year-old non-obese men, regardless of their physical fitness
level [43]. In contrast, in obese 40-year-old men, both in the LFL and HFL groups, after 10 WBC, the
CRP concentration was significantly lower than before the initiation of cryotherapy, which indicates
an anti-inflammatory effect of the treatments, and the concentration of IL-6, similar to that in young
men [43], did not change [26]. Many of the previously researches indicate the anti-inflammatory effect
of WBC [30]. Lubkowska et al. [59] found that in young men, the concentration of IL-6 in the blood is
higher than the baseline after both 1 WBC and 10 WBC exposures. However, in this research, the levels
of other markers of inflammation were not measured [59].
IL-6 has a pleiotropic effect and may play a pro-and anti-inflammatory role [60]. Diseases and
stress-related muscle damage cause a significant increase in the concentration of IL-6 in the blood,
with a simultaneous increase in the concentration of other pro-inflammatory cytokines, such as TNF-α
or IL-1β. In contrast, exercise that does not cause micro-damage increases the concentration of IL-6,
but does not increase the concentration of other pro-inflammatory cytokines [35]. In such a situation,
IL-6 shows a metabolic effect stimulating lipolysis and fat oxidation [34]. The persistently elevated
J. Clin. Med. 2020, 9, 2797 17 of 21

concentration of IL-6 in the blood indicates low-grade chronic inflammation, such as, for example, in
obese people [18,19]. It has been shown that visceral obesity is associated with high CRP and high
IL-6 levels [41]. But low levels of irisin, an anti-inflammatory effect, were found only in people with
high CRP, not high levels of IL-6 [41]. In our study we found a significant increase of the levels of
IL-6 in the blood of menopausal women with MetS in response to 10 and 20 WBC treatments, but
without a concomitant change in CRP levels, indicating the anti-inflammatory effect of IL-6 in this
situation. Unfortunately, we did not simultaneously study changes in the concentration of other
inflammation markers, such as other pro- and anti-inflamed interleukins, which limits the possibility
of unequivocal inference. Nonetheless, similar results were obtained by other researchers who, in
young healthy men, found higher than baseline levels of IL-6 in the blood after 5 WBC, 10 WBC and 20
WBC sessions [33]. These changes were accompanied by a simultaneous increase in the concentration
of anti-inflammatory IL-10 and a decrease in the concentration of pro-inflammatory IL-1α, without
significant changes in the levels of TNF-α, IL-1β and IL-12 [33]. In professional tennis players, it was
found that 10 WBC exposures used twice a day for 5 days in conjunction with moderate-intensity
training, the concentration of IL-6 in the blood increases, but at the same time, the concentration of
TNF-α decreases [61]. In turn, in obese men, the reaction to WBC was influenced by their fitness level.
In people with low fitness level, after 10 WBC sessions, there was a decrease in the concentration of
IL-6 and TNF-α, without affecting the change in people with high levels of fitness [25]. The results of
our study showed a beneficial relationship between the increase in IL-6 concentration and changes in
body composition in menopausal women with MetS. Showing statistically significant correlations, it
was found that a greater increase in IL-6 concentration was associated with a greater decrease in waist
circumference and a greater decrease in WHR and WHtR.
The results of our study indicate the stimulating effect of cryogenic temperatures on the secretion
of irisin and IL-6 and indirectly on their participation in the modulation of metabolic changes leading
to beneficial changes in body composition of menopausal women. However, the research has its
limitations. They mostly regard the assessment of changes in blood cytokine levels. This limits the
possibility of indicating the source of their secretion. Subsequent research should be focused on the
evaluation of mRNA expression for pro- and anti-inflammatory interleukins, as well as adipocytokines
and UCP-1 both in blood mononuclear cells, as well as in myocytes and adipocytes in response to
WBC treatments applied in subjects with varying degrees of fat and including both sexes. In our study,
we obtained significant changes as an effect of 20 WBC procedures, indicating the clinically beneficial
influence of these treatments on body composition (reduction of abdominal obesity) in menopausal
women. However, it seems that increasing the number of treatments would increase the clinical effects.
Considering the significant influence of changes in physical activity and diet on body composition, the
idea of our study was to exclude the influence of these factors and to check to what extent cryotherapy
treatments alone are able to induce beneficial changes in body composition among menopausal women.
The volunteers did not change their previous behaviour, nor did they participate in clinical therapy,
they were not subjected to pharmacotherapy, and did not change their physical activity or diet during
the trial. More research is needed including a greater number of volunteers that would undergo more
WBC treatments. Subsequent research should include people representing different levels of physical
activity and the researchers should simultaneously assess the effects of WBC and diet modification.

5. Conclusions
Whole-body cryotherapy, assuming the application of 20 treatments in the series, reduces
abdominal obesity in menopausal women indirectly through the secretion of irisin and IL-6, and can
be used as adjunctive therapy in the treatment of metabolic syndrome. Our conclusion is limited to
menopausal women with low-moderate physical activity for whom its level as well as diet were not
changed during the treatment.

Author Contributions: M.W., Conceptualisation, methodology, validation, formal analysis, investigation,

resources, data collection and storage, writing—original draft preparation, project administration. J.S. (Jadwiga
J. Clin. Med. 2020, 9, 2797 18 of 21

Szymura), formal analysis, investigation, resources, data collection and storage, writing—review and editing,
project administration. J.S. (Justyna Sproull), formal analysis, investigation, resources, data collection and storage,
writing—review and editing, funding acquisition. Z.S., Conceptualization, methodology, writing—review and
editing. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Science Centre, Poland, grant number 2014/2015/N/NZ7/03036,
and partly by University of Physical Education in Krakow, Poland, grant number 2016/BS/INB/20. Open Access
financed by University of Physical Education in Krakow, Poland.
Acknowledgments: We would like to thank all of the volunteers for their participation in this project. We thank
Jan Tabak for his assistance in the organisation and medical supervision during the application of whole-body
cryotherapy procedures at the Malopolska Cryotherapy Centre.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses or interpretation of data; in writing the manuscript or in the decision to publish
the results.

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