**2. Materials and Methods**

Twenty-five males of various fitness levels with minimum of one-year experience in resistance training recruited from colleges of physical education and through a website with readers interested in fitness and diets. Inclusion criteria were as follows: age between 18 and 30 years and a minimum one-year experience with resistance and aerobic training. Subject recruitment began in April 2019 and lasted until January 2020. Persons interested in participating were screened to ascertain they meet the minimum criteria for the enrollment into the study.

Exclusion criteria were current injuries or health conditions that might have affected sports performance or put them at risk for further injuries including the presence of cardiovascular diseases, diabetes mellitus, arterial hypertension, or any other diseases that required pharmacological treatment. Additionally, subjects taking any performance enhancing supplements (i.e., creatine, β-hydroxy β-methyl butyrate, caffeine, protein powder, weight gainer, thermogenics, etc.), were required to discontinue consumption at least one week prior to baseline testing and continue abstaining from their use for the remainder of the study. The study was approved (ethic approval code 764/18 S-IV)by the Human Ethics Review Board, First Faculty of Medicine and General University Hospital, Prague, Czech Republic and was performed in agreement with the principles of the Declaration of Helsinki as revised in 2008. Prior to randomization, all subjects were required to sign an informed consent.

Using electronic randomization system, subjects were randomly assigned to follow either a CKD or RD (both with total caloric intake reduction by 500 kcal/day) while participating in three strength workouts and three aerobic workouts per week (30 min run, heart rate around 130–140 beats/min.) for 8 weeks. Total caloric intake reduction by 500 kcal/day is counted from balanced hypocaloric diet with a reduction of energy intake by 500 to 1000 kcal from the usual caloric intake. The U.S. Food and Drug Administration (FDA) recommends such diets as the "standard treatment" for clinical trials (FDA, 1996)

Subject randomization and follow-up during the study is depicted in CONSORT diagram in Figure 1.

**Figure 1.** CONSORT diagram of subjects participating in an 8-week program while consuming a cyclical ketogenic reduction diet (CKD) or nutritionally balanced reduction diet (RD).

#### *2.1. Baseline and Postinterventional Testing*

Data collection during baseline and post-intervention testing included medical history, anthropometric examination, power performance test, bicycle spiroergometry, and blood drawings to obtain laboratory data. BMI was calculated by the scale, using the height measurement. Accurate height was measured using a basic stadiometer (Seca 222, Seca Co., London, UK).

#### 2.1.1. Biochemical and Anthropometric Examination

At baseline, all subjects were weighted, and their BMI was calculated. Body composition was measured using InBody Body Composition Analyzers (InBody230, InBody Co., Ltd., Seoul, Korea). Body weight and other body composition measurements (lean body mass, body fat mass, BMI, water content, and percentage of body fat) were taken with minimal clothes, no shoes, and measured to the nearest 0.5 kg.

Blood samples for biochemical measurements were taken prior to initiation of study and at the end of the study after 8 weeks of diet. Serum was obtained by centrifugation and samples were subsequently stored in aliquots at −80 ◦C until further analysis. The maximal storage time was 8 months.

Biochemical parameters liver test, urea, creatinine, and circulating lipids were measured to exclude liver, kidney, or lipid disorder. Creatine kinase and lactate dehydrogenase were measured to explore a possible influence of the diets on muscle regeneration. β-hydroxy-butyrate was measured to confirm a compliance to ketogenic diet.

β-hydroxy-butyrate was measured using TECOM Analytical Systems (TECOM Analytical Systems CS spol. s r.o., Prague, Czech Republic). Other biochemical parameters were measured by spectrophotometric methods using ARCHITECT c Systems device (Abbott Park, IL, USA.) in the Department of Biochemistry of the Institute for Clinical and Experimental Medicine in Prague.

## 2.1.2. Strength and Aerobic Performance Testing

Power and performance testing were conducted over a 5-day period. Subjects signed for an hour block to participate in each test. Each block had a maximum of 5 subjects in a gym and the spiroergometry was reserved for each of them for an hour. Subjects were instructed to arrive at the gym 30 min prior to testing times and not to train for at least 24 h before testing. A strength performance testing for power output in the three exercises—bench-press, lat pull-down, and leg-press was performed as follows: The subjects underwent an adequate warm up. After resting for two to four minutes the subjects than performed a one-repetition maximum attempt of each exercise with proper technique. If the lift/press was successful, after resting for another two to four minutes the load was increased by 5–10% and another lift/press was attempted. If the subject failed to perform the lift/press, after resting for two to four minutes they attempted the lift/press with weight reduced by 2.5–5%.

#### 2.1.3. Methodology of Strength Testing

Upon arrival, the primary researcher explained the testing procedures and protocols and demonstrated each test. Subjects were instructed to warm up. Power and aerobic performance test administrators and personal researchers were blinded to the randomized group allocations. Each proband participated in bench press, lat pull-down, and leg-press to assess the maximum power performance.

#### 2.1.4. Aerobic Performance Testing

Aerobic performance testing was carried out by bicycle spiroergometry using analyzer of respiratory gases (Quark CPET, 1850 Bates Ave, Concord, CA 94520, Cosmed, USA). This metabolic cart measures expired airflow by means of a pneumotach connected to the mouthpiece. A sample line is connected to the pneumotach from which air is continuously pumped to O2 and CO2 gas analyzers. Prior to testing, the pneumotach was calibrated with six samples from a 3 L calibration syringe. The gas analyzers were also calibrated before each test to room air and calibration gases (15.21% O2 and 5.52% CO2, respectively). Heart rate (HR) was continuously recorded during exercise by electrocardiography (Fukuda Denshi FX-8322 Cardimax ECG, 17725 N. E. 65th Street Bldg. C, Redmond, WA. 98052 USA).

Prior to exercise, the subjects were instructed to maintain a pedal cadence between 70 and 90 rpm during exercise and to exercise to volitional fatigue. We used a modified exercise step protocol 0.33 W.min−<sup>1</sup> as described by Gordon et al. [23]. The test was terminated when the subject was unable to maintain a pedaling cadence of 40 rpm.

Maximal oxygen consumption was assessed by the attainment of the following criteria: (1) a plateau (ΔVO2 ≤ 50 mL/min at VO2 peak and the closest neighboring data point) in VO2 with increases in external work, (2) maximal respiratory exchange ratio (RER) ≥ 1.10, and (3) maximal HR within 10 b/min of the age-predicted maximum (220-age). All subjects met the first two criteria.

Breath-by-breath gas exchange data from all tests were transferred to a spreadsheet program (MS Excel 365) for further analysis. In addition, data from the VO2max tests were time-averaged using 10 s intervals to examine the incidence of an oxygen plateau.
