The effects of a single and a series of Finnish sauna sessions on the immune response and HSP-70 levels in trained and untrained men

Abstract Background The aim of the study was to investigate the effect of a Finnish sauna on the immune status parameters. The hypothesis was that hyperthermia would improve immune system’s functioning by changing the proportion of lymphocyte subpopulations and would activate heat shock proteins. We assumed that the responses of trained and untrained subjects would be different. Material and methods Healthy men (20–25 years old) were divided into groups: the trained (T; n = 10), and the untrained group (U; n = 10). All participants were subjected to 10 baths (each one consisted of: 3 × 15-minute exposure with cooled down for 2 min. Body composition, anthropometric measurements, VO2 peak were measured before 1st sauna bath. Blood was collected before the 1st and 10th sauna bath, and 10 min after their completion to asses an acute and a chronic effect. Body mass, rectal temperature and heart rate (HR) were assessed in the same time points. The serum levels of cortisol, Il-6, HSP70 were measured with use of ELISA method, IgA, IgG and IgM by turbidimetry. White blood cells (WBC), leukocyte populations counts: neutrophils, lymphocytes, eosinophils, monocytes, and basophils were determined with use of flow cytometry as well as T-cell subpopulations. Results No differences were found in the increase in rectal temperature, cortisol and immunoglobulins between groups. In response to the 1st sauna bath, a greater increase in HR was observed in the U group. After the last one, the HR value was lower in the T group. The impact of sauna baths on WBC, CD56+, CD3+, CD8+, IgA, IgG and IgM was different in trained and untrained subjects’ responses. A positive correlation between the increase in cortisol concentrations and increase in internal temperatures after the 1st sauna was found in the T (r = 0.72) and U group (r = 0.77), between the increase in IL-6 and cortisol concentrations in the T group after the 1st treatment (r = 0.64), between the increase in IL-10 concentration and internal temperature (r = 0.75) and between the increase in IL-6 and IL-10 (r = 0.69) concentrations, also. Conclusions Sauna bathing can be a way to improve the immune response, but only when it is undertaken as a series of treatments.


Introduction
In athletes, as a result of intense physical training, the immune system is often suppressed [1].Thermal treatments, e.g., sessions in the Finnish sauna, can be used to support restitution after physical exercise [2].Different types of saunas are used.Dry sauna (Finnish sauna) treatments are most often used.Another type is the wet sauna (Russian bathhouse).Their effect on the human body varies.The high humidity of the air in a wet steam room makes it difficult for sweat to evaporate, which slows down the removal of heat from the body.A wet sauna causes a much greater heat load on the body, as shown by a greater increase in rectal temperature, a greater increase in heart rate, indicated intense subjective sensations, and a greater rate of physiological stress during wet sauna bathing [3,4].Previous studies have evaluated the influence saunas can have on the immune system.The effects reported include changes in the number of leukocytes [5,6], expression of pro and antiinflammatory interleukins [7,8], and reducing the risk of respiratory system diseases [9].
Overheating stresses the body and causes the increased production of interleukins and heat shock proteins (HSP) [10,11].However, the current research does not have a clear answer as to the reason for the increased amount of HSP in the plasma.The mechanisms may include increased intracellular expression and exocytosis of HSP, changes in the number of cells responsible for the production of HSP, or apoptosis or necrosis of these cells and release of their contents [12].According to Zychowska et al., the response to heat stress involves the increased production of the antiapoptotic HSP70 or the degradation of damaged HSP27 [13].Their research demonstrated that heat stressors induce the expression of genes encoding HSP, and mRNA levels for the HSP70 and HSP27 genes differed between athletes and nontraining participants.In the group of untrained men, the mRNA levels of all tested genes were higher in response to the same heat challenge.The authors concluded that the expression of genes related to heat-induced stress depends on the level of physical activity [13].
In the scientific literature, various protocols of repeated exposure to a thermal stimulus in the form of bathing in a sauna can be found [13][14][15][16].A popular protocol is to bathe for 1 h, 2 times a day for 7 d [14].However, in studies on the usefulness of the sauna as a factor improving post-exercise recovery, other protocols involving participation in treatments every 2 d are used [4,15,16].And such a protocol was also applied in this project.In order for the immune system reactions to fully manifest, 10 sessions in the sauna were performed.
The currently available literature supports that there are differences in the cellular response to the same stressors in trained and untrained subjects [5,13,17,18].Despite numerous studies on the body's response to bathing in a Finnish sauna, it is impossible to know whether the changes in plasma or blood cell markers are the result of changes in the plasma volume or changes in the protein-blood profile as a result of adaptation to heat stress.Improving the immune system functioning through exogenous stress is of great importance in a pandemic situation and may be an alternative for people with reduced immunity and an inability to exercise [19].Therefore, this study aimed to investigate the effects of a single session and a series of 10 baths in a Finnish sauna on the profile of white blood cells (WBC), the body's immune response, and the concentrations of selected interleukins, cortisol, and HSP70 in the blood of trained and untrained men.
Our hypothesis assumed that after the first sauna treatment, there would be an increase in the total number of WBCs.In the trained subjects, we expected that the number of neutrophils, basophils and lymphocytes would increase more than in untrained men.In untrained subjects, we expected greater changes in HSP-70 protein concentration after sauna treatments as a response to heat stress.We assume that in untrained people, the sauna can be a kind of substitute for physical training and its effect will be similar to the effect of physical exertion.Hence, we expect a strong reaction after the first bath and various adaptation processes after the 10th bath.For trained individuals, the thermal stimulus may have a lesser effect on the WBC, as these individuals' organisms are to some extent adapted to similar extrinsic stressors.

Study participants
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Bioethics Committee of the Regional Medical Chamber in Krakow (66/KBL/OIL/2011).
All participants gave written informed consent to participate in the study.This study included two groups of healthy males between the ages of 20-25.The training group (T; n ¼ 10) consisted of middle and long-distance runners with 5 ± 1.5 years of training and were members of the Academic Sports Association.The study took place between October and December (2013).All runners were in their detraining period and had last competed and/or trained 2 months prior to the study.Subjects from the T group were included in the project at the beginning of the 3rd week of this period.The detraining period is defined as follows: after 2 weeks of not performing trainings, in the 3rd-week aerobic training was introduced (3 times a week, moderate intensity).During detraining, the players did not use any supplements and did not undertake any biological regeneration treatments.The aerobic training performed by subjects from the T group was the standard procedure used during the deconditioning period.For each participant, a heart rate range corresponding to a moderate-intensity exercise was calculated from the measured VO 2 max and was set between 60 and 70% HRmax.Exercise intensity was monitored with a Polar Electro 610S heart rate monitor (Polar, Finland).Classes supervised by a qualified trainer were conducted on days when there were no sauna sessions.They were performed in an airconditioned room, and were general training exercises.The duration of a single training unit was 45 min.
The untrained group (N; n ¼ 10) consisted of volunteers, whose physical activity was estimated to be at the medium level (category 2) using the International Physical Activity Questionnaire (IPAQ) [20].The inclusion criteria for participation in the study included no abnormalities in baseline complete blood count (CBC) and electrocardiogram (ECG), absence of an acute infection, and no history of chronic diseases such as arterial hypertension, diabetes, and epilepsy.None of the study participants had attended a sauna or any another type of heat therapy sessions before participating in the study.The anthropometric characteristics of the study participants in both groups are listed in Table 1.There were no statistical differences in these parameters at baseline between the T and U groups.
Before participation in the study, all subjects were informed about the objective and methodology of the research project, the possible side effects, and the ability to resign from participation in the study at any time without stating a cause.The subjects were instructed to refrain from modifying their daily food intake, taking dietary supplements, and consuming alcohol during the entire study period.They were also asked for not taking any anti-inflammatory drugs during participation in the study.The diet of all participants was verified before participating in the project with the use of a standard diet diary in which they were recording food and beverage intake during 5 d.The content of micro-and macronutrients in the diets of all participants were within the normal ranges for the Polish population.

Experimental procedure
The study protocol consisted of a series of 10 exposures in a traditional Finnish (dry) sauna.Each exposure consisted of three 15-min sessions in the sauna chamber.Between each session, the body was cooled down for 2 min using running water at a temperature of approximately 20 C. The mean temperature in the sauna chamber at head level was 90 ± 2 C with a relative humidity of 5-16% [4].All sessions took place before noon on Mondays, Wednesdays and Fridays.The subjects fasted prior to each treatment for 12 h but were allowed to drink 0.5 L of water in the morning before treatment (not later than 30 min before entering the sauna).The subjects went into the sauna chamber without clothes and did not drink anything during the session.The intervals between treatments were 1 or 2 days (weekends) and all treatments were completed within 3 weeks.All treatments were supervised by a physician.The experimental procedure has been shown in Figure 1.

Anthropometric measures
Before the 1st and 10th treatments, the following parameters were measured: body weight (BW), body height (BH), heart rate (HR), and skinfold thickness.BW was measured using the F1505-DZA scale manufactured by Sartorius (Germany) which had an accuracy of up to 1 g.BH was measured with medical scales with an accuracy of 1 cm.HR was determined using the Sporttester Polar R400 (Finland).Skinfold thickness measurements were performed with a Harpenden skinfold caliper with 20 g pressure strength on the contact surface with an accuracy of up to 0.1 mm.The percentage of body fat (PF) was calculated using the formula described by Slaughter et al. [21].Before entering the sauna, rectal temperature sensors were placed 15 cm deep into the rectum.Rectal temperature (Tre) and HR were taken in 5-min intervals.The Tre [ C] was monitored with a CTD85M electrothermometer from Ellab, Radiometer (Denmark) with an accuracy of 0.1 C. HR was recorded using a Polar Elektro P-3000 heart rate monitor (Finland).A graded test until volitional exhaustion was performed on a cycloergometer (ER 900D, Jaeger, Hoechberg, Germany) during which peak oxygen consumption (VO 2 peak) was measured in the recruitment process, after verifying all the inclusion criteria for the project.

Blood analyses
Prior to and after completion of the 1st and 10th sauna baths, Ibefore the 1st sauna, IIafter the 1st sauna, IIIbefore the 10th sauna, and VIafter the 10th sauna, a venous blood draw was performed on each subject.Before the blood sampling, participants were asked to fast for 12 h Twelve milliliters were collected from each participant while in a sitting position and placed in three test tubes (Vacutainers: two with EDTA and one with a clotting activator).In the samples with EDTA, the total number of WBC with differential counts of neutrophils (NEUT), lymphocytes (LYMPH), eosinophils (EO), monocytes (MONO), and basophils (BASO) were determined.These analyses were performed by flow cytometry using a Sysmex XE 2100D laser hematology analyzer (Roche Diagnostics, USA).On the surface of LYMPH, the expression of the following cellular markers were determined: CD3 þ (T lymphocytes), CD4þ (Th helper lymphocytes), CD8 þ (cytotoxic lymphocytes, Tc), CD56 þ (NK cells), and CD19 þ (B lymphocytes).The analysis of LYMPH surface markers was performed by flow cytometry using fluorescently labeled monoclonal antibodies.The determination was performed with the Multitest CD3 FITC/CD16 CD56 PE/CD45 PerCP/CD19 APC and Multitest CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC antibodies from Beton Dickinson using the FACSCalibur flow cytometer (Becton Dickinson, USA).The results were analyzed using the CellQuest Pro software (Becton Dickinson, USA).Blood collected in the tubes containing a clot activator was centrifuged at 3500 rpm (1200 g) for 10 min at 4 C (MPW-351R, Med.Instruments, Poland).After centrifugation, serum, free from any traces of hemolysis, was evaluated for total protein using the biuret method on the Architekt ci8200 analyzer.Cortisol concentrations were determined using the enzyme-linked immunosorbent assay by DRG (DRG Instruments GmbH, Germany; sensitivity of 2.5 ng ml À1 ).The concentration of IL-6 and IL-10 cytokines were determined using the enzyme-linked immunosorbent assay by DRG (DRG Instruments GmbH, Germany; sensitivity for IL-6 0.03 pg ml À1 , for IL-10 0.05 pg ml À1 ).IgA, IgG, IgM immunoglobulins concentrations were determined using the turbidimetric method with the Architekt ci8200 analyzer (Abbott Diagnostic, USA).The concentration of HSP from the HSP-70 family was determined using the enzyme-linked immunosorbent assay (ELISA) by DRG (DRG Instruments GmbH, Germany), sensitivity 0.039 ng ml À1 ).
The change in plasma volume (% DPV) was calculated using the changes in total protein concentration determined before and after the selected sauna treatment sessions using the following formula [22]: Bpprotein concentration determined before the sauna bath, Bkprotein concentration determined after the sauna bath.
Due to the dynamic changes in plasma volume during sauna baths, the individual leukocyte fractions, lymphocyte subpopulations, and concentrations of cortisol, IL-6, IL-10, HSP-70, and immunoglobulins were corrected to reflect the changes in plasma volume.The corrected values were calculated using the formula described by Kraemer and Brown [23]: W ccorrected value, W bvalue measured before sauna bath, W avalue measured after a sauna bath.

Statistical analyses
Statistical analyses were carried out using Statistica 13.0 software developed by StatSoft (Poland).The normality of the distributions was assessed using the Shapiro-Wilk test.As the distribution of tested variables was not close to normal a non-parameter equivalent of one-way analysis of variance for repeated measurements (Friedman test) was used.For statistically significant results Conover post hoc test was calculated.In order to calculate the effect size, d Cohen (for parametric variables) and Kendall's W (for non-parametric variables) coefficients were calculated.Kendall's W uses Cohen's interpretation guidelines of 0.1 -<0.3 (small effect), 0.3 -<0.5 (moderate effect) and !0.5 (large effect).To assess the relationship between physiological and biochemical indices, Spearman's rank correlation coefficients were used.A significance level of p < .05 was assumed as statistically significant.All data are presented as the arithmetic mean values: x ̅ ± standard deviation (SD).

Systemic responses to sauna baths
Table 2 shows the changes in Tre after sauna baths.In both study groups, a statistically significant increase in Tre was observed after the 1st and 10th sauna sessions.Significantly lower initial temperature values were observed in the T group in response to a series of sauna baths (w ¼ 0.48).In response to a sauna bath, no significant differences were found in the increase in Tre between groups: T and U.
After the 1st and 10th heat baths, a statistically significant decrease in body mass (BM) was observed in both study groups (d ¼ 0.71 for T and 0.69 for U group).A greater difference in BM was noted after the 10th session compared to the 1st session in both groups (d ¼ 0.56 for T and 0.61 for U group).In response to sauna baths, no significant differences were found in BW loss between the two groups (Table 2).
In both study groups, a statistically significant increase in HR was found after the 1st and 10th sauna baths (d ¼ 0.86 for T and 0.89 for U group) (Table 2).After the last sauna bath, the HR value was significantly lower compared to the 1st bath in the T group (d ¼ 0.53).In response to the 1st sauna bath, a significantly greater increase in HR was observed in the U group than in the T group (d ¼ 0.49).
Sauna baths resulted in a statistically significant increase in cortisol concentration after the 1st (d ¼ 0.49 for T and 0.71 for U group) and 10th baths (d ¼ 0.44 for T and 0.65 for U group) in both study groups.The increase in cortisol concentration observed after completing 10 thermal exposures was lower compared to the 1st treatment (Table 2).In response to sauna baths, no significant differences in the increases in cortisol concentrations were found between groups.A statistically significant positive correlation between the increase in cortisol concentrations and the increase in internal temperatures after the 1st sauna was found in the T (r ¼ 0.72) and U group (r ¼ 0.77).
Table 3 shows the % DPV of study participants after sauna baths.After the 1st and 10th sauna baths, a decrease in plasma volume was observed in both groups.In both study groups, the mean loss of plasma volume was greater after the 10th bath compared to the 1st bath.The % DPV after the 10th bath was smaller in the U group compared to that observed in the T group, which was statistically significant (Figure 2).

Changes in immune response to sauna baths
After the 1st and 10th sauna baths, an increase in leukocyte count was observed in both study groups.However, only after the last sauna session did this change reach statistical significance in the T group (w ¼ 0.37).In response to the sauna baths, no significant differences were found in the WBC between the U and T groups after both the 1st and 10th heat exposures (Figure 3).Results could also be found in supplementary materials.There was a directly proportional relationship (r ¼ 0.74) between the increase in the total number of leukocytes and HR in the T group after the 1st sauna bath.
Changes in the number of cells from LYMPH subpopulations are shown in Figure 3. Statistically significant changes in the T group were found only in NK cells (CD56 þ ) between results obtained before the 1st and 10th sauna baths and before and after the 10th (w ¼ 0.48).For the U group, significant differences were shown for NK cells before and after the 1st exposure (w ¼ 0.37) and T (CD3 þ ) and Tc (CD8 þ ) LYMPH before and after the 10th treatment (w ¼ 0.34 and 0.32 respectively).There was also a significant difference in the number of B LYMPH (CD19 þ ) between samples drawn before the 1st and the 10th sessions (w ¼ 0.33).There was a significant difference between groups in B cell counts & -Significant differences at p < .05compared to the value after 1st sauna.# -Significant differences between T and U at p < .05.Table 3. Changes in plasma volume (% DPV) after the 1st and 10th sauna baths in trained (T) and untrained (U) men.
% DPV ( x ± SD) Group % DPV1 (before and after the 1st sauna bath) % DPV10 (before and after the 10st sauna bath) T À8.91 ± 3.85 À11.17  (CD19 þ ) after the 1st heat exposure (w ¼ 0.47).Results could also be found in supplementary materials.Figure 4 summarizes the measured immunological parameter mean values at each time point for both groups.Results could also be found in supplementary materials.A directly proportional positive relationship was demonstrated between the increase in IL-6 and cortisol concentrations in the T group after the 1st treatment (r ¼ 0.64).In the same group of men, after the 1st bath, a positive correlation was found between the increase in IL-10 concentration and internal temperature (r ¼ 0.75) and between the increase in IL-6 and IL-10 (r ¼ 0.69) concentrations.

Rectal temperature, plasma volume and physiological parameters
Despite a similar increase in Tre during the first and last sauna baths, the athletes showed greater efficiency in thermoregulatory mechanisms (greater sweat secretion and greater weight loss) which is consistent with the results of previous studies [24].This study demonstrates the beneficial effect of sports training, which enabled the athletes' bodies to respond more effectively to heat stress, as previously indicated by Pilch et al..The decrease in the observed resting temperature after a series of sauna treatments in the trained group indicates acclimation to the high temperatures.This finding was also observed by Bartolom e et al. in young semiprofessional football players who participated in 3 weeks of passive overheating in the sauna [25].The reduction in plasma volume was greater during the last bath in both groups, which is an adaptive effect.These changes were significantly higher in the T group compared to the U group.The greater degree of dehydration observed in athletes reflects the positive effect endurance training has on the body's adaptation to high temperatures and the functioning of the circulatory system [26].

Blood leukocyte counts
In the T group, a significant increase in the total number of leukocytes and NEUT was observed in response to both a single sauna bath as well as a series of 10 sessions.This finding was also observed in two additional studies [5,27].According to Shephard et al. [28], the mechanism responsible for leukocytosis after systemic hyperthermia is an increase in cardiac output and, consequently, an increase in leukocyte demargination.In our study, a positive correlation was observed between the increase in the total number of leukocytes and HR in the T group after the first sauna bath (r ¼ 0.74, p < .05).This is in support of the mechanism described by Shephard et al.Others have proposed alternative mechanisms which include increased expression of granulocyte colony-stimulating factor (G-CSF) [29] and the increased secretion of cortisol causing NEUT to migrate from the bone marrow into the bloodstream [30].In our study, there was an increase in cortisol levels after the 1st and 10th sauna baths.Similar results have been presented before [31,32], suggesting hypercortisolemia after baths could explain the leukocytosis.Activation of the adrenergic system in response to heat stressors increases cortisol levels [33].In both study groups, a lower increase in cortisol was observed in response to consecutive sauna sessions indicating thermal adaptation to similar thermal conditions [34].
The sauna baths caused a slight decrease in the number of T LYMPH (CD3þ) after the 1st and 10th treatment.However, a significant difference in the number of CD3 þ LYMPH was only noticed after the 10th bath in the untrained group. .Peripheral blood lymphocyte subpopulations expressed in absolute values before and after the 1st and 10th sauna baths in men from trained (T) and untrained (U) men.Ã Significant differences at the level of p <.05 compared to the value before the 1 st sauna.ÃÃ Significant differences at p <.05 compared to the value before the 10th sauna.&& Significant differences at the level of p <.05 compared to the value after 1st sauna.# Significant differences between T and U groups at p <.05.
The absolute number of Tc (CD8 þ ) decreased significantly only in the untrained men after the 10th treatment.There were no significant changes in the number of Th LYMPH (CD4 þ ) and B LYMPH (CD19 þ ) after both single and repeated sauna baths in both studied groups.The absolute number of NK cells (CD56 þ ) decreased immediately after the 1st and 10th sauna baths in Figure 4. Selected immunological markers expressed in absolute values before and after the 1st and 10th sauna baths in men from the trained (T) and untrained (U) men: (A) interleukins, (B)immunoglobulins, (C)HSP70.Ã Significant differences at the level of p < .05compared to the value before the 1st sauna; ÃÃ Significant differences at p < .05compared to the value before the 10th sauna; && Significant differences at the level of p < .05compared to the value after 1st sauna.
both study groups.A statistically significant change in the number of NK cells was noted in the U group after the 1st treatment and the T group after the 10th sauna bath.
The present study seems to confirm the direction of changes observed by Giannopoulos et al. [29] where 13 healthy volunteers participated in a one-time bath in a Finnish sauna.Researchers observed a negligible increase in the total number of leukocytes and NEUT.In our research, slightly different from previous observations [35], there were changes in the absolute number of T LYMPH (CD3 þ ).After the 1st and 10th bath in the sauna, a slight decrease in the absolute value of CD3 þ cells was observed, but only in the U group after 10 baths did this reach statistical significance.There were no significant changes in the absolute number of Th LYMPH (CD4 þ ).The absolute number of Tc (CD8 þ ) decreased slightly after the 1st and 10th baths but was only statistically significant after the 10th treatment in the U group.
The immune system's response to sauna bathing in trained and untrained men is similar to its activation during physical activity in athletes.Leukocytosis occurs after exercise and its elevation is directly proportional to the intensity and duration of exercise, and inversely proportional to the level of training [36].This is due to an increase in NEUT, LYMPH, and, to a lesser extent, MONO.Post-exercise EO counts decline while BASO levels do not change significantly [37].The lymphocytosis that occurs during and after exercise is associated with an increase in the sub-population of T LYMPH (CD4 þ and CD8 þ ), B LYMPH (CD19 þ ), and NK cells (CD56 þ ).NK cells increase faster than any other lymphocyte subpopulation.At the same time, the CD4 þ /CD8 þ cell ratio changes as the number of CD8 þ LYMPH increases faster than CD4 þ [38].

Cortisol and cytokines serum concentrations
The changes in cortisol levels caused by stress stimulation are associated with changes in cytokine and leukocyte levels.Glucocorticoids inhibit the expression of pro-inflammatory cytokines: IL-1, IL-2, IL-6, IL-8, IL-11, IL-12, TNFa, and INFc, while stimulating the production of the anti-inflammatory cytokines, IL-4 and IL-10.The reduction of pro-inflammatory cytokines is achieved by destabilizing and suppressing the transcription of mRNA [39].As reported in previous studies, passive exposure to heat causes an increase in the concentration of circulating IL-6 [40][41][42].
In our study, increases in IL-10 were recorded after the completion of the series of sauna baths and before the last treatment, but only in the T group was this change statistically significant.One can hypothesize that the adaptive increase in IL-10 was designed to limit the inflammatory reaction and production of pro-inflammatory cytokines after the applied hyperthermia.

Serum immunoglobulin concentrations
Little information is available on the effects of hyperthermia on serum immunoglobulin levels.In the research of Hietal et al. [43], the 1st and 10th heat baths increased the internal temperature by 1.2 C but did not produce any significant changes in the serum concentrations of immunoglobulins.In our study, after a series of ten sauna baths, there was an increase in the baseline values of immunoglobulins in both study groups, but only in the T group were these changes significant.This indicates a better and faster adaptation to difficult environmental conditions by athletes (increased plasticity of the athletes' immune system) [28].There was no correlation between immunoglobulin levels and the number of WBC, including B cells.

Hsp70 serum concentrations
The traditional Finnish sauna may support skeletal muscle hypertrophy by stimulating HSP70.This protein is involved in the structural development of skeletal muscle [44].It regulates muscle plasticity and acts as a molecular chaperone helping to refold or remove defective proteins [44,45].In this study, the influence of an increase in internal temperatures during sauna bathing on the concentration of HSP from the HSP70 family was determined.After bathing in the sauna, a statistically significant increase in the level of HSP70 was obtained in both studied groups with an increase in Tre of 1.47 ± 0.18 C in the T group and 1.61 ± 0.21 C in the U group.However, no correlation was found between the increase in HSP70 concentrations and the increase in Tre.After the 1st sauna bath, the serum concentration of HSP70 increased by 144% in the T group and by 271% in the U group compared to the level obtained before the bath.This highlights the fact that one-time heat treatments are a heavy burden on the body.However, repeated treatments (series of treatments) reduce the body's stress response [46] and thus should only be recommended in this capacity.After a series of 10 baths, a smaller increase in the concentration of HSP70 was found compared to the increase observed after the 1st treatment.The lower increase in HSP70 after the 10th session may be a result of the lower Tre experienced by the subjects after the 10th sauna.It was not possible to identify differences between changes in HSP70 concentrations in T and U groups due to large individual differences, but there was a tendency of a stronger reaction to heat stress in people from the U group.
It has been shown that a single exposure to heat in a sauna causes changes in the expression of genes encoding heat shock proteins in peripheral blood leukocytes.In the study by Bouchama et al., the expression of the gene responsible for the HSP-70 protein was significantly increased just after a 15-min exposure to the sauna [47].
In the study by Blatteau et al. [48], a thermal stimulus using an infrared sauna was applied and the concentrations of HSP70 in the serum were measured before and 30 min and 2, 8, and 24 h after the thermal exposure.An increase in serum HSP70 was only demonstrated 2 h after the end of the bath.The concentration of HSP70 in the present study, determined 10 min after the end of the sauna bath, increased significantly after the 1 st bath in both groups.
This may be explained by the higher increases in Tre than in the studies by Blatteau et al.

Study limitations
The study presented here has a number of limitations that should be taken into account when trying to interpolate the results to other populations.First of all, the research was carried out only on men, and as we know, both physical activity and thermal stimuli affect the bodies of women and men differently.Second, the study was conducted on young adults and the results will only apply to this group.Then, as trained persons, runners in the retraining process were observed, which influences the specificity of the results obtained.During the detraining period, the runners undertook aerobic activity.This factor may per se affect the biochemical results.As this project involved 10 sauna sessions over a 3-week period, he added that a comparison group of detrained runners not participating in sauna treatments would improve the findings of the study.The investigated markers may also change under the influence of diet and the amount of sun exposure, and these vary depending on the place of residence and the season of the year.Also, it should be mentioned that the stress reactions related with blood collection could activate the same genes as sauna heat exposure.Another important limitation of this study is the lack of a control group or control intervention.Such groups should be included in future studies.

Conclusions
In summary, a significant increase in WBC was only noted in the T group after a series of ten thermal baths in the sauna.This group also experienced an increase in IL-6 concentrations and a decrease in the number and percentage of CD56 þ cells.In the U group, a significant decrease in the number of CD3 þ and CD8 þ cells was noted.The results show a transient weakening of the nonspecific response in the T group (NK cells), and the specific immune response in the U group.A single sauna bath in both groups caused a significant increase in the concentrations of the cytokines IL-6 and IL-10 which may indicate the anti-inflammatory effect of hyperthermia on the human body.
The use of sauna baths can be a good solution for physically inactive people to improve specific cellular responses and increase the production of anti-inflammatory cytokines.However, such treatments should be recommended as a series.Sauna baths can also be a way for athletes to acclimate to high ambient temperatures.

Figure 2 .
Figure2.Peripheral blood leukocyte counts and their individual populations (x ± SD) before and after the 1st and 10th sauna baths in men from trained (T) and untrained (U) men.Ã Significant differences at the level of p < .05compared to the value before the 1st sauna.ÃÃ Significant differences at p < .05compared to the value before the 10th sauna.

Figure 3
Figure3.Peripheral blood lymphocyte subpopulations expressed in absolute values before and after the 1st and 10th sauna baths in men from trained (T) and untrained (U) men.Ã Significant differences at the level of p <.05 compared to the value before the 1 st sauna.ÃÃ Significant differences at p <.05 compared to the value before the 10th sauna.&& Significant differences at the level of p <.05 compared to the value after 1st sauna.# Significant differences between T and U groups at p <.05.

Table 1 .
Anthropometric characteristics and peak oxygen consumption of the studied men from trained (T) and untrained (U) groups [x mean ± SD].

Table 2 .
Changes in rectal temperature (Tre), body mass (BM), heart rate (HR) and cortisol concentration after the 1st and 10th sauna baths in trained (T) and untrained (U) men.Significant differences at the level of p < .05compared to the value before the 1st sauna.ÃÃ -Significant differences at p < .05compared to the value before the 10th sauna.