*2.1. Subjects*

Forty-one highly trained athletes (28 men, 13 women), ages ranging from 18 to 35 years, participated in the study. They were members of the Polish national team or athletes taking part in national and international competitions. They represented the following sport disciplines: triathlon (Olympic distance: 1.5 km swim, 40 km bike ride, 10 km run) (seven men, four women); long-distance running (5 km, 10 km, and marathon) (six men, two women); Olympic taekwondo (six men, one woman); sprint (100 m, 200 m, and 4 × 100 m relay) (six men, one woman); canoeing (three men); and fencing (five women). Before starting the study, each participant was informed about the aim and procedures, potential risks, and the possibility to withdraw at any time without giving any reason. All athletes gave their written consent to participate in the examinations and fulfilled a questionnaire on their health status and potential contraindications. All athletes had valid health certificates issued by a physician who specialized in sports medicine, thus were eligible for training and competition. Exclusion criteria were illness symptoms, injuries, and taking drugs (temporarily or chronically). Only the data of those athletes who were present at both examinations was analyzed. The study was conducted in accordance

with the Declaration of Helsinki. The Ethics Committee of the Poznan University of Medical Sciences in Poland approved the study protocol (approval no. 1017/16 issued on 5 October 2016).

#### *2.2. Training Characteristics*

All participants attended training sessions at least six times a week. During the whole 7-week period under study (general preparation phase of the one-year cycle), the athletes had on average 57 training sessions of a total duration of 71.2 h. The average duration of a single session was 84 min.

#### *2.3. Study Design*

The study was conducted in the Human Movement Laboratory of the Department of Athletics, Strength and Conditioning at the Poznan University of Physical Education (Pozna ´n, Poland). Athletes arrived at the laboratory in the morning. During all measurements, the constant temperature was maintained (20-21 ◦C) by an air conditioning system. On the day of the examination, the participants could only eat a light breakfast. It was also recommended for them to avoid co ffee or tea for 12 h, alcohol for 24 h, and hard exercise for 48 h before each examination. After arriving, athletes changed into their lightweight sports clothing (without watches and wristbands potentially a ffecting blood flow) and acclimatized to the laboratory conditions for at least 30 min. During this time, they completed the required questionnaires, and height and weight measurements were performed.

Athletes underwent the examinations twice: at the beginning of the general preparation phase and after seven weeks, at the end of this phase. Each time, the same procedure was applied: (1) initial resting blood pressure measurement; (2) resting NADH fluorescence measurement; (3) blood draw, (4) incremental exercise test; (5) second blood draw; (6) post-exercise blood pressure measurement; and (7) post-exercise NADH fluorescence measurement (3 min after the end of the test).

#### *2.4. Incremental Exercise Test*

The exercise test was conducted on the H/P Cosmos treadmill (h/p/cosmos sports & medical GmbH, Nussdorf – Traunstein, Germany). All participants were familiar with the treadmill test because they regularly (2-3 times a year) participated in similar tests. The purpose of this examination was to assess maximal oxygen uptake (VO2max) and peak heart rate (HR).

Respiratory gases were collected and analyzed using the MetaMax 3B ergospirometer (Cortex Biophysik BmbH, Leipzig, Germany) and the MetaSoft Studio 5.1.0 software (Cortex Biophysik BmbH, Leipzig, Germany). The exercise protocol started with a 4-min warm-up at the treadmill speed of 6 km/h. Then, the treadmill accelerated by 2 km/h every 3 min. The treadmill inclination was 1% throughout the whole test. The test terminated if the athlete signaled his/her voluntary exhaustion by raising one hand. Maximal oxygen uptake was considered to be reached if the oxygen uptake (VO2) was stabilized despite the further increase in treadmill speed. All participants were highly trained, so during the test, all of them reached a plateau in VO2 uptake. We also checked three additional conditions to confirm reached maximal oxygen uptake: (i) HR reached at least 95% of the age-adjusted HR; (ii) cuto ff blood lactate concentration ≥ 9 mmol/L for man and ≥7 mmol/L for women; and (iii) respiratory exchange ratio was ≥1.1 [28]. Heart rate was measured using the Polar H6 Bluetooth Smart monitor (Polar Electro Oy, Kempele, Finland) attached to a chest strap.

#### *2.5. Lactic Acid Measurements*

Capillary blood samples were obtained from the fingertip at rest and 2 min after the exercise test. A total of 20 μL of whole blood was drawn to a micro test tube using a capillary. Biosen C-line (EKF Diagnostics, Cardi ff, UK) was used to measure the level of lactate.

#### *2.6. Anthropometric Measure*

Anthropometric measurements were performed according to standardized procedures. Body mass (kg) and height (cm) were measured with a digital measuring station Seca 285 (SECA, Hamburg, Germany). Body mass index (BMI) was calculated as body weight divided by height squared (kg/m2).

#### *2.7. Nicotinamide Adenine Dinucleotide Fluorescence*

NADH fluorescence was measured using the AngioExpert device (Angionica, Łód´z, 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 cu ff 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 (cu ff 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):


**Figure 1.** Parameters describing the Flow Mediated Skin Fluorescence. Bmean—Mean value of the basal fluorescence; FImax—Maximal fluorescence during ischemia; FRmin—The first minimal fluorescence value during reperfusion; Imax—The net increase in fluorescence over the baseline during ischemia; IRampl—The amplitude of fluorescence change during ischemia and reperfusion; Rmin—The net reduction in fluorescence below the baseline. Reprinted from Bugaj 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.

**Figure 2.** A sample Flow Mediated Skin Fluorescence measurement in a 23-year-old male sprinter. Changes in nicotinamide adenine dinucleotide fluorescence are shown before and after 7-weeks of training, at rest, and after cardiopulmonary exercise test until exhaustion. The first 2 min serve to determine the baseline fluorescence level. This was followed by a 200-s ischemia (increase in fluorescence) and a 290-s reperfusion (decrease in fluorescence).
