## 2 Materials and Methods
### Nicotinamide Adenine Dinucleotide Fluorescence  
NADH fluorescence was measured using the AngioExpert device (Angionica, Lodz, Poland, 2016) based on the flow mediated skin fluorescence (FMSF) method. FMSF enables recording of the changes in NADH fluorescence as a function of time in response to ischemia and reperfusion in forearm skin cells. During the measurement, AngioExpert emits light at the wavelength of 460 nm [6; 7]. NADH molecules have autofluorescence capability at a wavelength of 460 nm [9]. The changes in fluorescence intensity observed during the examination are produced in the most superficial skin cells (epidermis) [6; 29], which is due to very shallow skin penetration by excitation light at the wavelength of 340 nm. About 90% of the recorded signal comes from the skin depth up to 0.5 mm. The activated skin region is not directly supplied with blood, but is supplied with oxygen by deeper blood vessels [6; 7; 29].  
During the examination, each participant sat on a chair with his/her arm resting on the measuring device. Immediately before examination, systolic (SBP) and diastolic (DBP) blood pressure was measured using the Omron 3 (Omron, Kyoto, Japan) device. At the start of the FMSF examination, basal fluorescence was registered for 2 min. Then, an occlusion cuff was inflated up to the pressure of 50 mmHg above the SBP for 200 s. After this time, blood flow in the forearm was restored (cuff deflated) and the changes in NAD fluorescence were recorded for a further 3 min [7].  
The following parameters related to NAD fluorescence were measured or calculated (Figure 1):  
* B\({}_{\text{mean}}\)--Basal fluorescence at the wavelength of 460 nm, recorded at rest at the beginning of the measurement;
* FI\({}_{\text{max}}\)--The maximal increase in fluorescence above the baseline observed during forearm ischemia;
* FR\({}_{\text{min}}\)--The maximal drop in fluorescence below the baseline observed during reperfusion;
* I\({}_{\text{max}}\)--The relative increase in fluorescence = the difference between I\({}_{\text{max}}\) and B\({}_{\text{mean}}\);
* R\({}_{\text{min}}\)--The relative drop in fluorescence = the difference between B\({}_{\text{mean}}\) and FR\({}_{\text{mean}}\);
* IR\({}_{\text{ampl}}\)--The maximal range of changes in fluorescence = the sum of R\({}_{\text{min}}\) and I\({}_{\text{max}}\); and
* CI\({}_{\text{max}}\)--The relative (percentage) contribution of I\({}_{\text{max}}\) to IR\({}_{\text{ampl}}\)[7].  
Figure 1: Parameters describing the Flow Mediated Skin Fluorescence. B\({}_{\text{mean}}\)—Mean value of the basal fluorescence; FI\({}_{\text{max}}\)—Maximal fluorescence during ischemia; FR\({}_{\text{min}}\)—The first minimal fluorescence value during reperfusion; I\({}_{\text{max}}\)—The net increase in fluorescence over the baseline during ischemia; IR\({}_{\text{ampl}}\)—The amplitude of fluorescence change during ischemia and reperfusion; R\({}_{\text{min}}\)—The net reduction in fluorescence below the baseline. Reprinted from [PERSON] et al. [5].  
The second measurement was made according to the same methodology, 3 min after the end of the treadmill test. A sample measurement of the NADH fluorescence from a 23-year-old male sprinter before and after training was shown in Figure 2.