2.3. Laboratory Tests

The subjects were instructed to refrain from consuming caffeine and alcohol and from heavy training on the day before the tests, as well as to consume a light meal at least 3 h before each laboratory test. During the 3-h period preceding the tests, only ad libitum water ingestion was permitted. During all laboratory tests, HR was collected via a HR monitor (HRM-Tri; Garmin Ltd., Olathe, KS, USA) across the chest with sampling at 1 Hz. Body mass was measured with a body fat monitor scale (TBF-102; Tanita, Tokyo, Japan) before the cycling and running tests.

The subjects completed a two-part test consisting of a submaximal intermittent test and a maximal incremental test that were performed in a swimming flume at a constant water temperature of 25.8 ± 0.8 °C. The subjects wore their own technical trisuits, standard swimming caps, and goggles throughout the incremental swimming test. The submaximal intermittent test was performed first. The swimming speed of the submaximal intermittent test was individualized according to the average swimming speed of the most recent 1500-m time trial (S₁₅₀₀) of each triathlete. The speed for the initial stage was 70% of S₁₅₀₀ and increased by 5% at each subsequent stage for a total five to seven stages, each consisting of 4 min of exercise and 2 min of rest. Before the test and after each stage, blood samples were obtained from the fingertip and the BLa was measured with a lactate analyzer (Lactate Pro 2; Arkray, Inc., Kyoto, Japan). The submaximal intermittent test was concluded when the BLa exceeded 4.0 mmol·L⁻¹ or the rate of perceived exertion was ≥15. Following a 5-min recovery period after the submaximal intermittent test, the maximal incremental test was performed. The initial speed was set at that of the next to last stage of the submaximal intermittent test and then was increased by 0.03 m·s⁻¹ every minute until volitional exhaustion, which was defined as the point at which the subject could no longer swim at the required speed.

The maximal incremental test was performed on an electronically braked, indoor cycle trainer (CompuTrainer Pro; RacerMate Inc., Seattle, WA, USA), which allowed the subjects to use their own bicycles, at a constant room temperature of 25.2 ± 1.2 °C, relative humidity of 40.4% ± 8.3%, and barometric pressure of 755.4 ± 3.5 mmHg with an electric fan ensuring air circulation around the participant. The maximal incremental test was performed following a 5-min warm-up period (100 W) and a 5-min recovery period. The initial workload was set at 100 W and then increased by 20 W·min⁻¹ and cadence was maintained at 80 or 90 rpm in accordance with race cadence of the individual until volitional exhaustion, which was defined as <75 or 85 rpm (i.e., a decrease of 5 rpm as compared with the cadence of the sustained during test) continuously for 5 s. To determine the PO during the test, which was used for analysis, the bicycles were fitted with calibrated power measuring pedals (Garmin Ltd.) at a sampling rate of 1 Hz.

The maximal incremental test was performed on a motorized treadmill (ORK-7000; Ohtake-Root Kogyo Co., Ltd., Iwate, Japan) at a grade of 1% to accurately reflect the energetic cost of outdoor running [33] following a 5-min warm-up period (9.0 km·h⁻¹) and a 5-min recovery period. The experimental environmental conditions were similar to those of the incremental cycling test. The initial speed was set at 9.0 km·h⁻¹ and then increased by 0.6 km·h⁻¹ every minute until volitional exhaustion, which was defined as the inability of the subject to continue running at the required speed. The treadmill belt speed, which was used for analysis, was measured with a hand-held tachometer (EE-1B; Nidec-Shimpo Corporation, Kyoto, Japan).