## 4 Discussion
### The Effect 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  
Figure 4: Calculated parameters. Flow Mediated Skin Fluorescence parameters in athletes (N = 41) in two examinations, before and after the cardiopulmonaryanory exercise test. I\({}_{\max}\)—Changes in the net increase in fluorescence over the baseline during ischemia; IR\({}_{\text{ampl}}\)—Changes in the amplitude of fluorescence change during ischemia and reperfusion; R\({}_{\text{min}}\)—Changes in the net reduction in fluorescence below the baseline; CI\({}_{\max}\) – Changes in I\({}_{\max}\)/IR\({}_{\text{ampl}}\) ratio. Values are means (SD). A two-way analysis of variance (relation between exercise and training), post-hoc Scheffe test, significant differences between pre- and post-exercise: *** \(p<0.001\), *** \(p<0.01\), * \(p<0.05\); significant differences between before and after training * \(p<0.001\), * \(p<0.01\), * \(p<0.05\). 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\({}^{+}\) and NADH are in balance (i.e., the more NAD\({}^{+}\), 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. VO\({}_{2}\)max 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 (B\({}_{\text{mean}}\), FI\({}_{\text{max}}\), FR\({}_{\text{min}}\)). The post-training increase in B\({}_{\text{mean}}\), FI\({}_{\text{max}}\), and FR\({}_{\text{min}}\) 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].