To the best of our knowledge, this is the first study assessing the effect of WBC on vitamin D status. We found significant changes in 25(OH)D levels in both groups 30 minutes after 10 days of cryotherapy, compared to pre-therapy levels (Table 2). We found an increase in the HPhL group and a decrease in the LPhL group. We also observed a further slight decrease in the serum 25(OH)D level 24 hours after the tenth WBC stimulus compared to the pre-therapy values, exclusively in the LPhL group (Table 2). It should be noted that one might expect a small increase in 25(OH)D concentration during the few days in the middle of June in which the research was conducted, because, as observed Osmancevic et al.28, it takes approximately 7 days for serum 25(OH)D levels to peak after ultraviolet B exposure. As Poland has a latitude of 49–54°N, the solar angle and weather conditions suitable for vitamin D synthesis occur between late April and early September29. Some studies have indicated that the exposed skin area and the baseline vitamin D status also affect the synthesis of cholecalciferol in the skin. Osmancevic et al.28 found that exposure of smaller body surface areas, such as the face and hands (while study participants are wearing a T-shirt and shorts), induces less vitamin D production. Moreover, other studies showed that current sun-exposure practices of the general population do not provide sufficient amounts of vitamin D30,31.

Despite not finding any studies on the effect of cryotherapy on the level of vitamin D in the available literature, we presume that the observed changes in 25(OH)D levels might be attributed to the role of vitamin D in the inflammatory response, which is indicated by the inverse relationship between changes (Δ 1–2 ) in concentrations of 25(OH)D and hsCRP, noted 30 minutes after the first WBC treatment in the HPhL group (Table 3). Studies have shown that vitamin D may possess natural antioxidant and anti-inflammatory properties24,32,33.

Moreover, our study shows that a series of 10 WBC sessions contributed to diverse systemic thermogenic responses depending on the level of physical fitness of the study participants.

We found an increase in serum irisin after 10 days of therapy compared to the pre-therapy level (Table 2), which was not statistically significant (17% in the HPhL group and 16% in the LPhL group). In the LPhL group, there was a significant increase 24 hours after the tenth application compared to the level observed before the application of WBC (Fig. 3).

Figure 3 Experiment schedule WBC = whole-body cryotherapy = blood collection = before first WBC, 2 = 30 minutes after first WBC, 3 = 24 h after first WBC, 4 = before tenth WBC, 5 = 30 minutes after tenth WBC, and 6 = 24 h after tenth WBC. Full size image

Our results are partly comparable to those reported by Dulian et al.34, who found a 20% increase in the plasma irisin concentrations in middle-aged, obese, non-active men and a slight decrease in active subjects 24 hours after 10 WBC sessions. The authors suggested that an increase in the level of irisin following exposure to a low ambient temperature may be induced by shivering thermogenesis, which causes irisin secretion from skeletal muscle. However, the inverse relationship between the muscle mass and irisin concentration obtained by Dulian et al.34 may point to the subcutaneous adipose tissue as the source of irisin.

Moreno-Navarrete et al.35 and Roca-Rivada et al.36 also showed that irisin was secreted not only from the muscles, but also from the visceral and subcutaneous adipose tissue. No such correlations were found in our study; however, it must be noted that only lean men were enrolled in our study, whereas the authors cited above conducted their studies on individuals with an elevated body fat percentage.

In our study, we evaluated the effects of WBC treatments on the level of Mst, which plays an important role in regulating energy homeostasis through the modulation of skeletal muscle mass37. Previous research findings suggested that inhibition of the TGF-β/Mst superfamily increases the activity of BAT, which leads to increased energy expenditure and provides metabolic benefits21.

Our results showed significant changes in Mst concentration only in the LPhL group 24 hours after the tenth WBC session compared to the values found 30 minutes after completing these session (Fig. 3). However, Mst concentration did not differ significantly from pre-therapy values. Our results are consistent with that of the available literature stating that changes in the Mst levels induced by environmental stimuli and/or physical exercise are transient and return to the baseline level within 24 hours after the application of the triggering factor38. In turn, the activation of satellite cells inducing inflammation requires more than 24 hours39.

In the LPhL group, we found positive correlations between changes in levels of 25(OH)D and Mst 30 minutes after the tenth WBC treatment (Δ 4–5 ) and compared to baseline (Δ 1–5 ) (Table 3). These results, as well as the negative correlations between changes (Δ 4–5 ) in concentrations of 25(OH)D and irisin in the HPhL group (Table 3), confirmed the role of vitamin D in skeletal muscle metabolism. Slivka et al.40 found that the induction of peroxisome proliferator-activated receptor (PPAR)-γ Coactivator (PGC-1α) gene expression is enhanced after exercise in a cold environment. Irisin is secreted in response to PGC-1α17, which may act as a co-activator of the vitamin D receptor in mitochondria41. Furthermore, Garcia et al.42 showed that vitamin D suppresses the expression of Mst, while it up-regulates the expression of follistatin and insulin-like growth factor II.

A considerable number of studies point to the anti-inflammatory effect of WBC1,3,43,44; therefore, we evaluated the levels of hsCRP and IL-6. In recent years, studies have indicated that Mst stimulates IL-6 production in muscle cells and adipose tissue45; however, in our study, we did not find a significant relationship between these molecules. It should be noted that in our study, blood was collected after physical exercise, which was performed immediately after WBC treatment. As shown by Rhind et al.46, there is a lower expression of genes encoding for proinflammatory cytokines in young healthy volunteers immediately after the training preceded by exposure to low temperatures, compared to training alone.

In the HPhL group, a significant change in the concentration of IL-6 was noted only after the first WBC stimulus (Fig. 2). No statistically significant changes after 10 days of cryotherapy were found. Our findings are consistent with the results previously published by Lubkowska et al.47, who showed that a series of WBC sessions did not trigger an increase in the concentration of proinflammatory cytokines in healthy young men. On the other hand, Ziemann et al.48 found a decrease in tumour necrosis factor alpha with a concurrent increase in IL-6 after 5 days of treatments conducted twice a day in conjunction with moderate-intensity training in professional athletes. This finding was explained by the fact that WBC significantly lowered the inflammatory response induced by eccentric exercise. The inverse relationship between changes (Δ 1–2 and Δ 1–6 ) in the concentration of irisin and IL-6, noted by us in the HPhL group (Table 3), points to the anti-inflammatory characteristics of irisin, which is consistent with the findings of Mazur-Biały et al.49.

Our study has some potential limitations. In particular, a small number of subjects participated in this study. Additionally, the study period was too short to observe the long-term effects of WBC. There was also a lack of standardised treatment protocols with regard to temperature ranges, timing, and frequency of exposure to WBC, which is likely to elicit varying recovery responses to the therapy.

Our data demonstrated that in healthy young men with a normal body weight, cryotherapy affects levels of 25(OH)D. The body’s response to a series of 10 cryotherapy treatments is modified by the physical fitness level. It should be noted that the observed changes in the serum 25(OH)D were small and transient, therefore athletes can use WBC treatments. However, in order to fully explain the body’s response to cryostimulation, additional studies need to be performed on a larger study group.