To evaluate the immunomodulatory effect of AMR supplementation, cytokine and cytometry parameters of immunological balance were analyzed in a group of rowers from the National Polish Team who were adapted to a very intensive training and exceptionally high lactic acid levels as a result (Table 3). It was presumed that the supplement intake would maintain the immunological balance, disordered by fatigue involved in both bouts of acute exertion and the long duration of intensive camp training. The research was conducted with seemingly small groups of participants that constitute actually a large part of the population of elite rowers, selected in terms of physical performance and training experience, similar age and body build, the same operating environment, a similar lifestyle, and diet. This strict selection minimized the influence of these factors on the analysis result. The consistency of the results achieved thanks to this made it possible to consider them as reliable. Perhaps, if the physiological models of changes in components extracted from the tissues in response to exertion were known, it could be possible to obtain more accurate results, with the use of based on these models indices of change. However, the more general indicator of relative change used in the research (RC) allowed a reliable analysis.
The immuno‑stabilizing effect of AMR was visible in the stabilization of base (initial) levels of numerous parameters, along with the shift of the immunological balance towards Th1 response after the second exertion test, crowning the six‑week supplementation period. Our main observation was that in the supplemented subgroup, long‑term training did not cause visible changes in the NK [CD3/CD16+/CD56+] initial percentage, in contrast to the placebo subgroup, for whom the percentage clearly decreased (RC = ‑65 [‑104 ‑25]%). Furthermore, Tδγ levels increased (RC = 45 [7 83]%), while the Treg percentage did not change significantly, which consequently decreased the value of the Treg/Tδγ ratio, relative to the control group (RC = 51.98 [‑113.07 9.12]%, d = 0.86). Analogically, relative to the control group, the long‑term training camp also resulted in a post‑recovery (I‑R) increase in IL2/IL10 ratio (RC = 48.28 [18.05 78.51], d = 1.61) as a consequence of post‑exertion (I‑E) IL2 decrease (RC = -23.2 [-41.02 -5.37], d = 1.33).
After the camp, an increase in the initial levels of IL2, IL10, CTL, and Tδγ with a decrease in IL2/IL10 ratio and IL4 were also observed, thus suggests a general activation of the immune system . During the second examination, the initial (I) CTL [CD8+/TCRαβ+] percentage increased to a similar degree both in the supplemented (RC=68 [25 111]%) and the control (RC = 64 [34 94]%) subgroup – the greatest observed among all analyzed cell populations. This reveals the impact of the long term intensive training itself on enhancing the number of CTL cells, which was not modifiable by AMR supplementation. On the other hand, NK notably decreased in the placebo subgroup, while in the supplemented subgroup, their average level remained at the baseline. This could be considered a protective effect of the AMR supplementation on NK levels, preventing their decrease under the impact of Treg and strenuous exercise. Similarly, Tδγ lymphocytes increased in both subgroups (RC = 44.70 [6.70 82.71]% supplemented and 22.64 [‑4.58 49.87]% placebo).
CTL, NK, and Tδγ cells are the WBC subsets representative of the Th1, cell‑mediated immune response. They are responsible for cytotoxicity, which is known to be a powerful stressor able to shift the Th1/Th2 balance towards the Th2, but it may have been impaired due to strenuous exercise . As observed in our study, the effects of AMR supplementation on NK and Tδγ cells were influential, helping sustain the balance between the cytotoxic lymphocytes and the Treg. The I‑I difference in initial Treg lymphocyte counts as well as Treg/Tδγ ratios between the first and second examinations increased more significantly in the control than in the supplemented subgroup. This observation means that AMR may prevent the cellular immunological response from weighting in favor of the Th2 response in circumstances of strenuous exercise stimulation.
Despite the beneficial Th1 shift observed in cytometry of supplemented athletes, their IL2/IL10 ratio presented relatively lower values, characteristic of Th2 response. The lowering of the post‑camp initial (I‑I) IL2/IL10 ratio in both subgroups indicates a shift towards Th2 response (Figure 1); this index was lower in the supplemented subgroup than in control, which is a consequence of the lack of a clear increase of initial IL2, accompanied by increase of IL10. During the recovery period after exertion, however, when athletes are the most susceptible to URTI due to immunosuppression , the supplemented athletes attained higher values of both IL2 and IL2/IL10 ratio with respect to control group, which further demonstrates the beneficial immune stabilizing actions of AMR. The after recovery increase in IL2/IL10 ratio in the supplemented subgroup observed after the second examination occurred by an experimental differentiation of IL2’s reaction to exertion which in turn shaped a converse differentiation of the changes, thus restoring the balance during rest (I‑R). This reaction was strong enough to be mirrored in the similar I‑R changes in IL2/IL10 ratio.
During the second examination, after the camp, the post‑exertion I‑E decrease in IL2 was greater in the supplemented group in respect to the controls; in the control subgroup, the decrease was less significant as compared to the first examination as a result of elevated post‑camp initial IL2 values. In the supplemented subgroup, this enabled a reproduction of post‑exertion IL2 levels similar to the values from the first examination, followed by an adequately fast return to the baseline during rest, resulting in a larger I‑R increase that suggests greater dynamics of changes in IL2 levels. Even though I‑E values of IL2 after the camp decreased more in the supplemented subgroup and less in the control, it did not modify the I‑E, but rather the I‑R values for IL2/IL10 ratio according to the reports about the connection between the two. Post‑recovery IL10 change trends seem to reproduce the post‑exertion IL2 change trends; while IL2 in the supplemented subgroup decreased after exertion, IL10 decreased during recovery, which, along with the I‑R IL2 increase, resulted in elevation of IL2/IL10 ratio, thus indicating a shift towards Th1 response.
The post‑exertion decrease observed in IL2 levels, followed by its increase back to the baseline, was noted in both examinations; in the second examination, however, said decrease was greater in the supplemented subgroup, as compared to both the control subgroup and the results of the first examination. Such a set of changes suggests a transient shift in immunological balance towards Th2 response resulting from exertion, followed by restoration of Th1 response intensity, which was reduced in placebo subgroup and sustained in the supplemented subgroup after long‑term training.
A previous study  revealed a crucial role of IL2 in the efficient suppression of T effector cells by Tregs. Since Tregs themselves do not synthesize IL2, their function depends on levels of this cytokine paracrinally released by other particular cells. The results suggest that Treg compete for IL2 with T effector cells, suppressing their function by consumption of this crucial cytokine. Therefore, even though high doses of IL2 make T effector cells immune to suppression by Treg, certain levels of this cytokine are crucial for Treg immunosuppressive function and priming their IL10 synthesis. This hypothesis seems to be in line with data obtained from our study, and it would explain why, as compared to the supplemented subgroup, the control was characterized by a greater post‑camp I‑I increase in IL2, a cytokine mainly described as typical of Th1 response, while the immunological balance of the counts of assessed cells seemed to be shifted towards Th2 response. It could also explain the relation between IL2 and IL10 cytokines: if IL2 primes IL10 synthesis by Treg, then lower post‑exertion IL2 levels in the supplemented subgroup could contribute to a post‑recovery decrease in IL10.
Another manifestation of sustained immunological balance was also immunosuppression prevention, as observed through a lower Treg/Tδγ ratio in the supplemented subgroup. Therefore, the post‑exertion shift towards Th2 response had a different character in both subgroups: in the control subgroup it was clearly visible, as seen in level changes of immunologically active cells, while it was mostly observed in cytokine changes in the supplemented subgroup. This indicates that AMR has adaptogenic effects, expressed as a stabilization of immunological parameters, along with the maintenance of essential levels of Th1‑related cytotoxicity in reaction to exertion despite the general tendency towards a Th2 response.
Our study on AMR intake in athletes demonstrates a beneficial shift of Th1/Th2 balance that is in line with existing articles on the use of this supplement. Although it was observed on a basis of different indices, compositional observations are partially convergent. Captured among our observations was a larger resting increase in IL2/IL10 ratio along with an increase in IL2, a cytokine typical of Th1 response; this is consistent with an earlier study, which reported an increase in IL2 in AMR supplemented rats subjected to forced swimming and food restriction , but it opposes a study on asthma model in mice, in which Th1/Th2 change resulted from a decrease in Th2 response cytokines .
In this study, the supplement did not modify the IL4 or IFN‑ɣ reaction, which has been reported by other authors [17, 21]. The diversity of immune response models and stressors  could explain this; in our case, for example, we noted that a clear lack of changes in IFN‑ɣ is typical of strenuous exercise . We also observed that the cytokine profile depends presumably on the load type, as numerous other works indicate . Changes in other blood parameters have also been presented in articles concerning exertion . Reduced blood lactic acid levels were noted in the supplemented athletes, which is in line with animal studies on AMR intake [60, 61].
According to past reports [13, 14], AMR is a non‑toxic, bioactive substance that exhibits a relevant immunomodulatory potential, and its beneficial effects on athletes were confirmed in our experiment. However, further research administration using greater doses of this supplement should be considered. It would also be useful to extend the panel of diagnostic tests by certain cytokines not involved in this introductory paper (e.g. TNF, IL17, IL1, IL6) to expand knowledge regarding the supplement’s active mechanisms.