Effect of Post-Exercise Sauna Bathing on Targeted Gut Microbiota and Intestinal Barrier Function in Healthy Men: A Randomized Controlled Trial

Background: Body temperature uctuations induced by acute exercise bouts may also inuence the intestinal barrier with related effects on epithelial permeability, immune responses, and release of metabolites produced by the gut microbiota.To evaluate the combined inuence of 4-weeks exercise training and post-exercise sauna treatments on gut bacteria and fungi, inammation, and intestinal barrier function. Methods: Randomized, parallel group design with pre- and post-study measurements. Fifteen (15) untrained males aged 22±1.5 years were randomly assigned to exercise training (ET) with or without post-exercise sauna treatments (S). Participants in the group ET+S (n=8) exercised 60 minutes, 3 times per week, on a bicycle ergometer followed by a 30-minute dry Finish sauna treatment. The control group (ET, n=7) engaged in the same exercise training program without the sauna treatments. Blood and stool samples were collected before and after the 4-week training program. Blood samples were analysed for the concentration of high-sensitivity C-reactive protein (hsCRP) and complete blood counts. Stool samples were analysed for pH, quantitative and qualitative measures of targeted bacteria and fungi, zonulin, and secretory immunoglobulin A. Group comparisons showed no signicant differences for blood and stool sample measurements during the 4-week study. Results: Group comparisons showed no signicant differences for blood and stool sample measurements during the 4-week study. Conclusions: The combination of 4-weeks exercise training and post-exercise sauna treatments did not have a measurable inuence on targeted gut microbiota, intestinal barrier function, and inammation biomarkers in young males compared to exercise training alone.

. These data imply that thermal therapies such as sauna bathing may in uence the gut microbiome.
To date, there is little information on whether thermal therapy exerts positive or negative effects on the microbiome. This is an interesting question because of the immunological effects of thermal therapies, and the potential interactive effects with exercise training.
Taken together, both exercise training and sauna bathing have the potential to in uence gut permeability, in ammation, and the gut microbiota. The aim of this study was to evaluate the effects of post-exercise sauna bathing in young men undergoing endurance training on the targeted gut microbiota and intestinal barrier function.

Participants
Study participants included 15 young healthy men aged 22 ± 1.5 years. The study inclusion criteria included voluntary written consent, absence of medical contraindications, no history of infections, and no injuries in the last 4 weeks prior to the study. Exclusion criteria including the intake of antibiotics, steroids, oral antifungal agents (except for topical antifungals), antiparasitic agents, pre-and/or probiotics, history of travel to tropical countries during the last 4 weeks before the study, and history of adverse responses to sauna bathing.

Procedure
This study utilized a randomized, parallel group design. The participants were randomly assigned to exercise training (ET) with or without post-exercise sauna treatments (S). Participants in the group ET + S (n = 8) exercised 60 minutes, 3 times per week, on a bicycle ergometer followed by a 30-minute dry sauna treatment. The control group (ET, n = 7) engaged in the same exercise training program without the sauna treatments. The 60-minute exercise bouts were performed on calibrated Keiser M3 ergometers (Germany).
The initial exercise intensity was set at 50% VO 2 peak for 2 weeks, and then increased to 60% for the nal two weeks of training. The physical exercise was performed in controlled environmental conditions (temperature of 22-23°C, and relative humidity of 30-33%) with no use of fans or cold drinks.
Immediately after nishing the 60-minute exercise bout, subjects from group ET + S spent 30 minutes in a dry sauna (in the sitting position), at a temperature of approximately 90°C at the chest level and relative humidity of air 10 ± 2%. The sauna treatment was divided into two or three parts (e.g. 3 x 10 minutes, 2 x 15 minutes), and subjects were allowed to cool the body for a maximum of 3-min (e.g. by taking a cold shower, immersing the body in cool water up to the armpits).
Participants agreed to maintain normal dietary intake patterns, and this was veri ed with 3-day food records at the beginning and end of the 4-week study. Energy and nutrient intake was calculated using the NUVERO application.

Peak oxygen uptake
Blood and stool sample collection Blood and stool samples were collected before and after the 4-week study. Blood samples (approx. 2 ml) were taken from the antecubital vein and centrifuged at 4000 rpm and 4°C. The serum was separated from the sample and stored at − 70°C. The concentration of high-sensitivity C-reactive protein (hsCRP) was measured by immunoenzymatic assay using a commercially available kit (DRG International Inc., Spring eld Township, NJ, USA; test sensitivity: 0.1 mg/L and 5 ng/mL). Complete blood count indices were determined by ow cytometry with a Synergy 2 SIAFRT analyser (Bio Tek, Winooski, VT, USA).
In order to perform qualitative analyses of selected indicator bacteria in the gastrointestinal tract, and to determine the stool pH, the studied men were requested to provide a stool sample within 24 hours of collection. Stool sample collection was performed according to the established protocol developed by KyberKompaktPRO (Institute of Microecology). To this end, a 150-ml sterile container was to be lled to three quarters of its volume with material preferably taken from eight different locations, and closed tightly with a lid. The indicator bacteria, sIgA (marker of mucosal immunity), and the concentrations of zonulin (marker of intestinal permeability) in stool were evaluated before and after completing the training programme in both studied groups of men.
Bacterial DNA was isolated from stool samples using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Danish). An appropriate quantity of stool was weighed into a sterile tube. The isolation of bacterial DNA from the stool sample was performed according to the manufacturer's protocol. The DNA eluates were stored frozen until subsequent analyses.
The anaerobic bacteria including Faecalibacterium prausnitzi of the genus Faecalibacterium, Akkermansia muciniphila of the genus Akkermansia, Bi dobacterium spp. of the genus Actinobacteria, and Bacteroides spp. of the genus Bacteroidetes were determined by Real-Time PCR with appropriate primers (ThermoFisher Scienti c, USA) ( Table 2). The reaction mixture contained QuantiFast SYBR Green PCR Kit (Qiagen), RNase-free water (Qiagen), and a mixture of forward and reverse primers selected for the bacteria tested. The analyses were conducted in an ABI 7300 analyser (ThermoFisher Scienti c, USA).
Bacterial DNA was isolated from a stool sample using the QIAamp Fast DNA Stool Mini Kit (QIAGEN). To this end, an appropriate quantity of stool was weighed into a sterile tube. The isolation of bacterial DNA from a stool sample was performed according to the manufacturer's protocol. The DNA eluates were stored frozen until subsequent analyses. The counts of anaerobic bacteria including Faecalibacterium prausnitzi of the genus Faecalibacterium, Akkermansia muciniphila of the genus Akkermansia, Bi dobacterium spp. of the genus Actinobacteria, and Bacteroides spp. of the genus Bacteroidetes were determined by Real-Time PCR with appropriate primers (ThermoFisher Scienti c) listed in Table 1. The analyses were conducted in an ABI 7300 analyser (ThermoFisher Scienti c). The nal bacterial count/g of stool was obtained by converting the number of copies of the sequence ampli ed by PCR in the bacterial genome (for Faecalibacterium, Akkermansia muciniphila, Bi dobacterium spp., and Bacteroides spp., respectively) and the dilution factor applicable to the kit used for DNA isolation from stool samples. The conversion factor employed in the study was checked and validated at the Institute of Microecology in Herborn, Germany. Table 1 presents the standards used in the studies.
The limit of detection for the evaluated parameters was 10 2 [CFU/g of feces]. For values below 10 2 [CFU/g of feces] [cut-off point], the value of 0 was adopted for statistical analysis, which, however, does not mean that the test sample was bacteria-free. The results of quantitative bacterial analysis were converted to the decimal logarithm (Log10). The entire Real-Time PCR methodology was developed and validated by the Institute of Microecology in Herborn, Germany. Reference values for selected indicator bacteria and stool pH are presented in Table 2.

Statistical analysis
The results are depicted as mean values, standard deviations (± SD). The data were analyzed statistically using the Statistica 13.0 software package (StatSoft, Tulsa, Oklahoma, USA). The normality of the variables was veri ed with the W Shapiro-Wilk test. The signi cance of differences between the variables observed before and after the training period in both study groups was assessed by the Wilcoxon matched-pairs test. The signi cance of differences between the groups was assessed by the nonparametric U Mann-Whitney test, with p < 0.05 adopted as the threshold for statistical signi cance.

Results
There were no signi cant differences between the two groups for age, body height, body mass, and percentage of body fat. The changes in VO 2peak did not differ between the groups during the 4-week study (Table 3). Group changes during the 4-week study did not differ signi cantly for targeted stool bacteria (Table 4). Group changes during the 4-week study did not differ signi cantly for targeted stool bacteria and stool pH (Table 5). Group changes during the 4-week study did not differ signi cantly for stool zonulin, sIgA, and hsCRP (Table 6). Group changes during the 4-week study did not differ signi cantly for total blood white blood cell (WBC) counts or WBC subsets (Table 7).

Discussion
In this study, participants exercised 60 minutes for three times per week over a 4-week period with or without 30-minute post-exercise dry sauna treatments. Blood and stool samples were collected pre-and post-study. The key nding of the present study is that repeated applications of sauna bathing just after physical training had no effect on targeted gut microbiota, intestinal permeability markers, intestinal barrier function, and in ammation in young adult males.
Microbial diversity can be altered by a variety of environmental factors including diet, altitude, season, temperature, and tness status (11). And conversely, changes in microbial diversity can in uence energy metabolism, behaviour, in ammation, immunity, aging, other physiological processes, and disease states (12).
This study did not include a non-exercise control group and was therefore not designed to investigate exercise training effects on targeted gut microbiota, intestinal permeability, or in ammation. Instead the primary focus was on the in uence of post-exercise sauna bathing on gut microbiota during a 4-week exercise training period. The results were not supportive that 30-minute sauna treatments after 60-minute exercise bouts altered levels of targeted bacteria from the gut microbiome.
Animal studies support that remodelling of the gut microbiome is responsive to chronic alterations in both ambient and internal temperature (13); (14), but human data, especially within the context of repeated acute thermal treatments are lacking (10); (15). For example, 24-week-old female mice exposed to 34°C for 8 weeks experienced a signi cant alteration in microbial composition, with increases in several genera including Akkermansia (14). The mechanism by which thermal therapy affects intestinal microbes may in part be related to temperature-related in uences on intestinal permeability and related effects on gut-derived metabolites (10). A single layer of epithelial cells connected by tight junctions forms the intestinal barrier that controls transports of metabolites from the lumen to the circulation. With increased body temperature, blood ow to the skin is increased accompanied by vasonconstriction of the GI tract, exerting tensional stress on tight junction and enhancing leakiness of selected metabolites.
Intensive and prolonged exercise can also induce intestinal hypoperfusion, dehydration, impaired osmolarity of body uids and gut motility, and increased permeability of the intestinal barrier (16);(17); (18); (19). Fecal zonulin is a recognized biomarker of intestinal permeability (17). In our study, an increase in zonulin concentration in feces was noted in both groups, but these increases did not differ between groups indicating no added effect of post-exercise sauna bathing. Microbiota imbalances such as low counts for Akkermansia muciniphila and Faecalibacterium prausnitzii have been linked to in ammation (20). The fecal pH level was within the normal range for our subjects, and re ects typical levels of shortchain fatty acid production by gut bacteria (7). Fecal abundance of A. muciniphila and F. prausnitzii may increase in response to physical training, but our data did not con rm these ndings (21); (22).
Secretory IgA plays an important role in mucosal immunity, can survive in harsh environments such as the GI, and provides rst-line protection against potentially pathogenic microbes (23).Gut sIgA imbalances have been lined to various diseases (24). Lifestyle, exercise, stress, and diet can in uence sIgA levels (25). Study participants had fecal sIgA levels within the reference range and changes during the 4-week study did not differ between ET + S and ET groups. Enterococcus spp., Lactobacillus spp. and Bacteroides spp. may have an in uence on intestinal in ammation by increasing the expression of tight junction protein and the production and secretion of mucin and antimicrobial peptides (AMPs) that combat pathogenic invasion and stimulate plasmocytes for IgA secretion (26). Our data indicate that 4weeks of exercise training with or without sauna bathing has no in uence on sIgA secretion by GI epithelial cells. Additionally, no group differences were observed for measures of in ammation including blood leukocyte subset counts and hsCRP. These data indicate that repeated sessions of post-exercise sauna bathing had no effect on pro le of the targeted gut bacteria, immune barrier function or in ammation markers in young males.

Limitation Of The Study
This study had several limitations. A randomized parallel group design was employed, and subject numbers were low for this type of investigation. However, the selected sample size was big enough to detect changes in the study's primary and secondary outcomes. The exercise training program and sauna treatments included just 12 sessions during a 4-week program, and this may not have been a su cient physiological stimulus to induce change in the measured outcomes, especially in young adult males.
Leukocyte subset and hsCRP levels were low pre-and post-study for both groups, indicating that there was little room for exercise training or sauna treatments to exert a measurable effect.

Conclusion
The combination of 4-weeks exercise training and post-exercise sauna treatments did not have a measurable in uence on targeted gut microbiota, intestinal barrier function, and in ammation biomarkers in young males.
This nding does not preclude other bene ts linked to exercise training and sauna treatments.
Selected indicators of gut microbiota remained stable during the 4-week exercise training period.

Abbreviations
AMPs antimicrobial peptides; VO2peak:peak oxygen uptake, GI:gastrointestinal; hsCRP:high-sensitivity Creactive protein; NS:non-signi cant difference; sIgA:secretory immunoglobulin A; WBC:total blood white blood cell Declarations Ethical approval The study protocol was approved by the Ethics Committee for Human Research at the Poznań University of Medical Sciences (approval no. 173/16 of 4 February 2018) and was performed in accordance with the Declaration of Helsinki. Males provided informed consent.
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Availability of data and materials
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.