**4. Discussion**

In this study, for the first time, the changes in NADH fluorescence in epidermal cells have been investigated in highly trained athletes before and after a training period. The main and novel finding is a significant increase in NADH fluorescence after training.

#### *4.1. The E*ff*ect of Training*

In our study, an increase in NADH fluorescence after a 7-week training period in highly trained athletes was observed. It is widely known that physical training induces several adaptations including mitochondrial adaptations [22]. The measurement of NADH fluorescence may be used to indirectly evaluate the mitochondrial function and information about its metabolic status [1,5]. However, the changes in NADH fluorescence alone do not allow us to answer the question of what particular metabolic changes took place. It is known that NAD<sup>+</sup> and NADH are in balance (i.e., the more NAD<sup>+</sup>, the less NADH and vice versa [8]). Therefore, the higher post-training NADH fluorescence shown in our study may indicate increased NAD turnover.

Our participants represented different sport disciplines, but the study was only conducted in the general preparation period of the annual training cycle. The main goal of this period, regardless of sports discipline, was the development of endurance capacity. VO2max did not change after training in our athletes, which is in line with other reports [30,31] that also did not observe such changes in highly trained athletes in an annual training cycle. However, we assume that the changes occurred at the cellular respiration level. The endurance-dominant training in all athletes significantly affected the increase in the NADH fluorescence, which can be reflected by the changes in mitochondrial functions as shown in measured NADH parameters (Bmean, FImax, FRmin). The post-training increase in Bmean, FImax, and FRmin suggests a training-induced increase in the total NAD pool. However, there is a lack of research on training-induced changes in skin mitochondria. We can only compare our findings with those obtained from muscle mitochondria. To the best of our knowledge, the only research on training-related changes in NAD levels was performed on rat muscles. It has been found that NAD levels increased in response to endurance training [16]. There is a lack of studies on NAD changes in trained humans. The training-related changes in mitochondria have been widely described in human muscles [19,21,22,25,32]. The training-related changes in the mitochondria are probably connected with the improvement in mitochondrial biogenesis and the removal of dysfunctional mitochondria [21,22,25,32]. After training, an increase was observed in the levels of proteins related to mitochondrial biogenesis [21,25] and an improvement in mitochondrial respiratory function [19]. It is suggested that the profile of the mitochondrial changes depends on training intensity and volume. Training volume seems to affect mitochondrial content, whereas training intensity is correlated with the improvement in mitochondrial respiration [19]. It must be remembered that exercise does not necessarily imply exactly the same metabolic changes in muscle and skin mitochondria. However, intense physical activity affects mitochondrial activity and induces an increase in NADH fluorescence, which we have shown in our previous study [5]. Therefore, the observed increase in NADH fluorescence after 7-weeks of training may indirectly indicate adaptive changes in skin mitochondria.

#### *4.2. Exercise Response*

In our recent paper [5], we showed that a single bout of exercise until exhaustion induced a significant increase in skin NADH fluorescence. The results of this study are in line with our previous observations. We found that the Imax parameter, related to fluorescence intensity, decreased after exercise and that the Rmin parameter increased after exercise. The likely explanation is that with limited aerobic metabolism, NADH is accumulated and the NAD+ amount decreases because anaerobic metabolism does not allow for restoring NAD+ from NADH to a sufficient extent [33–35].

However, some authors [36] sugges<sup>t</sup> that the decrease in NADH fluorescence intensity during reperfusion not only shows the change in mitochondrial function, but also in microcirculatory and endothelial functions related to the efficiency of blood supply to the skin. Both the skin blood vessels' thermoregulatory [37–39] and endothelial [40] functions improved after training. Our study supports this view and suggests improvements in exercise tolerance based on NADH fluorescence measurement.

#### *4.3. Practical Application*

The FMSF method might be useful to evaluate metabolic adaptations related to mitochondrial function and/or microcirculatory function as the effect of training (training efficiency). This might also be used to observe the recovery after exercise when returning to the resting NADH values.
