**2. Methodology**

#### *2.1. Selection of Participants*

A total of 26 male cyclists (11 PRO, 15 AMA) were recruited and completed the study. The PRO were competing at the *Union Cycliste Internationale* (UCI) PRO TOUR level and have participated in UCI major stage races (*Vuelta a España, Giro d'Italia, Tour de France*). The 15 AMA were from the southeast region of Spain. The PRO riders were selected based on the following criteria: (1) 20 to 40 years of age, (2) enrolled in a professional licensed team and (3) competed in at least one of the main 3-week stage races in the last years. Subjects for the AMA group had to meet the following inclusion criteria: (1) 20 to 40 years of age, (2) had at least 3 years of cycling experience and (3) performed specific training 6–12 h/week.

All subjects signed the informed consent document before their participation. The study was performed following the guidelines of the Helsinki Declaration for Human Research [19] and was approved by the Ethics Committee of the Catholic University of Murcia (CE091802).

#### *2.2. Study Protocol*

The experimental design of the study required each rider to visit the laboratory twice between the end of October and December (i.e., post-season period). In the first visit, a medical exam and blood analysis were completed to check their state of health. In the second visit (post-48 h), the cyclists performed a maximal incremental test. The 2 h prior to this latter test, they ingested a standardized breakfast, which was based relative to body mass (557.7 kcal) and composed of 95.2 g of carbohydrates (68%), 19.0 g of protein (14%) and 11.3 g of lipids (18%), established by a sports nutritionist. All subjects were instructed to refrain from high-intensity training 48 h before each visit.

#### *2.3. Incremental Test*

An incremental step test with final ramp until exhaustion was performed on a cycle ergometer (Cyclus 2TM, RBM elektronik- automation GmbH, Germany) using a metabolic cart (Metalyzer 3B. Leipzig, Germany) to determine VT1, VT2 and VO2max, as well as the associated levels of power output. The testing protocol started with 35 W and increased by 35 W every 2 min until RER > 1.05 was reached, from which the final ramp (+35 W·min<sup>−</sup>1) until exhaustion was initiated [20]. To ensure that VO2MAX was achieved, at least 2 of the following criteria had to be met: plateau in the final VO2 values (increase ≤ 2.0 mL·kg−1·min−<sup>1</sup> in the two last loads), maximal theoretical HR (220-age)·0.95) [21], RER ≥ 1.15 and lactate ≥ 8.0 mmol·L−<sup>1</sup> [22,23]. Ventilatory thresholds were obtained using the ventilatory equivalents method described by Wasserman [24].
