*2.2. Experimental Design*

Regarding design, this was a double-blind, crossover, and counterbalanced study to evaluate the influence of 2 weeks of betaine supplementation on apoptosis, oxidative stress, and aerobic capacity after EEE in a healthy male population. The participants were asked to consume betaine or placebo, with at least 3 weeks of washout period between the trials. All participants performed a graded exercise test (GXT) until exhaustion on the treadmill to determine VO2max before supplementation [31], and the speed equivalent to 80% VO2max was implemented for an EEE test conducted on a treadmill for 30 min, after which speed was increased by 0.2 m/s every 1 min until exhaustion. The participants were instructed to consume a standard breakfast consisting of 648 kcal of total energy (66% carbohydrate, 11% protein, and 23% fat) 1 h before exercise. Blood samples were drawn before exercise (Pre), immediately after (Post-0), and 3 h after (Post-3) the EEE to determine the biomarkers of apoptosis and oxidative stress. Furthermore, plasma concentrations of betaine and choline before supplementation and at the Pre time point of EEE (after two weeks of supplementation) were also analyzed. In addition, the time to exhaustion, peak oxygen consumption, maximal heart rate (HRmax), and average heart rate (AHR) were recorded during the exercise period. Figure 1 illustrates the scheme of the study.

**Figure 1.** Experimental scheme. GXT, graded exercise test; EEE, exhaustive endurance exercise; Pre, before exercise; Post-0, immediately after exercise; Post-3, 3 h after exercise.

#### *2.3. Supplementation Protocol*

The betaine supplementation strategy in the present study was based on the one suggested by Apicella et al. [23], wherein all participants consumed either 1.25 g of betaine (betaine powder; Twinlab, CO, USA) mixed with 300 mL sports beverage (Pocari Sweat, Otsuka, Taipei, Taiwan) or placebo (only sports beverage) twice per day for 2 weeks. The betaine for the supplementation was extracted from natural sugar beet and subsequently purified (99% pure). The sports beverage contains carbohydrates (66 g/L) and electrolytes of sodium (21 mEq/L), chloride (16 mEq/L), and potassium (4.9 mEq/L). The supplements for both trials had the same color and taste. The participants consumed supplements after breakfast and dinner during the experiment period.
