**1. Introduction**

Skin microcirculatory function and efficiency of blood supply to the skin can impact mitochondrial activity and the changes of nicotinamide adenine dinucleotide (NADH) fluorescence in the reduced form [1]. Mitochondrial function can be indirectly evaluated by NADH fluorescence [1] that has been measured in animals [2,3] and humans [1,4] at rest and under various conditions (e.g., ischemia and temperature changes). Bugaj et al. [5] were the first to describe the time course of NADH changes in the skin in athletes at rest and after exercise. In their study, a new method of evaluating NADH fluorescence—flow mediated skin fluorescence (FMSF)—was utilized. The FMSF method is based on the ability of NADH to autofluorescence. The fluorescence measured using the FMSF method reflects the dynamics of in vivo changes in NADH levels in most superficial layers of the skin [5–7]. Bugaj et al. [5] have shown that exercise to exhaustion induces changes in skin NADH fluorescence, in other words, the values recorded after exercise were higher than those before exercise (increase in: basal NADH fluorescence 13%, maximal NADH fluorescence 7% and minimal NADH fluorescence 12%).

Nicotinamide adenine dinucleotide (NAD) is synthesized in the cytosol, mitochondria, and nucleus. This molecule is active in the cytoplasm during glycolysis and in the mitochondria during oxidative phosphorylation when adenosine-5 -triphosphate (ATP) is produced [8]. NAD occurs in two forms: oxidized NAD+ and reduced NADH. NAD takes part in many biological reactions including electron transport. The reduction of NAD<sup>+</sup> to NADH occurs almost exclusively in the mitochondria at the final stage of cellular respiration [9,10].

In the human body, there is a pool of NAD that takes reduced (NAD+) and oxidized (NADH) forms, transforming into each other [8]. Importantly, the NAD pool is only constant for relatively short periods [8,11]. In the long term, the NAD amount changes depending on several factors such as age, diet, physical activity, medicaments, boosters, time of the day, etc. [11]. NAD+ metabolism is complex and includes many NAD+-consuming pathways as well as de novo and salvage pathways [8].

Mayevsky and Barbiro-Michaely [1] have claimed that the monitoring of the NADH level in tissue provides important information about the mitochondrial metabolic state (energy production, amount of intracellular oxygen). In addition, changes in the NAD+/NADH ratio reflect cellular respiration processes in mitochondria, thus indirectly represent their function [1,5]. Studies on changes in NADH in response to physical exercise were performed on animal and human skeletal muscle samples, but not in the skin [8,9,12]. Early reports including animals did not provide a clear answer as to how NADH levels were modified by exercise [13,14]. Subsequent human research had shown that intensive exercise, unlike light exercise, shifted the NAD+/NADH balance toward NADH [8,15]. Only Koltai et al. [16] have examined the influence of endurance training on changes in NAD+ level in rat muscles and showed that training resulted in an increase in NAD+ biosynthesis.

Studies on skeletal muscle mitochondria are valuable, but usually invasive due to the use of the biopsy technique [17,18] and expensive if transmission electron microscopy is used [19]. However, it has been suggested that physical exercise brings beneficial changes not only in skeletal muscle mitochondria, but also in skin mitochondria [20]. It has been demonstrated that physical exercise results in several beneficial mitochondrial adaptations [19,21–25]. Various changes were extensively studied in skeletal muscle mitochondria [19,21,25–27], while only one study dealt with the changes in the skin [20]. However, we do not know whether training only a ffects muscle mitochondria, or the adaptations also take place in skin mitochondria that are easily accessible to study because they lie superficially.

To the best of our knowledge, there is a lack of studies describing the e ffect of physical training on changes in NADH fluorescence in the skin. The novel, noninvasive, and cheap flow mediated skin fluorescence method can be a source of valuable information about the mitochondrial activity. Therefore, the study aimed to evaluate the changes in NADH fluorescence in the superficial skin layer resulting from a 7-week training period in highly trained competitive athletes. We hypothesize that physical training results in an increase in the NADH fluorescence levels in athletes.

#### **2. Materials and Methods**
