Detailed descriptions of the study protocol have been published elsewhere [3]. Briefly, our ten male subjects’ average values in age, height, body weight, body mass index and VO2max were 24.0 ± 3.3 (range: 20–31) years, 170.8 ± 5.0 (162.1 - 175.6) cm, 61.9 ± 5.7 (54.6 - 69.7) kg, 21.2 ± 1.7 (18.4 - 23.3) kg·m-2, and 3.18 ± 0.45 (2.34 - 3.75) L·min-1 (or 52.0 ± 9.2 (36.2 - 61.4) ml·kg-1·min-1), respectively. The aim and design of this study were explained to every subject before each gave their written, informed consent. This study was conducted in accordance with the guidelines proposed in the Declaration of Helsinki. The Ethical Committee of both JAXA (reference number: 32-2-7) and University of Tsukuba (22–283) reviewed and approved the study protocol.
The subjects completed three experimental sessions separated by approximately 1 week. During each session, the subjects performed one of three exercise trials, i.e., SIT, HIAT, or CAT. The detailed protocols of the three cycling exercises are shown in Additional file 1: Table S1. All aspects of the experimental session remained the same during all three sessions except the actual exercise technique. The three sessions were implemented in random order.
On each session day, the subjects arrived at our laboratory at approximately 7:15 am. They were asked to minimize any physical activity (walking etc.) while en route from their home to the laboratory. Six subjects drove their own cars to the laboratory, and 4 subjects were picked up at their homes and driven to the building by a research assistant because they did not have their own cars. At 7:45 am, a subject would seat himself in a comfortable armchair and remain in a resting (seated) position without movement until 8:20 am. From 8:05 to 8:20 am (15 minutes), the subject was connected to a face mask of the indirect calorimeter for baseline data collection. Average VO2 data during the final 10 minutes were used as the baseline value. After the baseline measurements, we disconnected the face mask and allowed the subject to take a drink of water before riding on the cycling ergometer (75XL Ш, Konami, Tokyo, Japan) for the exercise phase. After the water, he was reconnected to the indirect calorimeter and started pedaling the ergometer at 8:25 am.
After the exercise phase, the subject returned to the resting position immediately but stayed connected to the indirect calorimeter for 10 minutes to measure immediate post-exercise VO2. After 10 minutes, we removed the face mask but the subject remained in the resting position for another 170 minutes. During the post-exercise phase, the subject remained in the resting position immobile. Ten minutes of VO2 measurements were started at 30, 60, 90, 120, 150, and 180 minutes post exercise. For each measurement session, average VO2 data during the final 5 minutes was used.
All gas exchange measurements were measured using the mixing chamber method with an open-circuit computerized indirect calorimeter (AE-310S, Minato Medical Science, Osaka, Japan). The ventilatory volume, VO2, and VCO2 were calculated every 15 seconds. The gas analyzer was calibrated before each trial. Coefficient of variation (CV) from the mean for the three resting VO2 measurements in the 10 subjects was 4.3%.
To measure each subject’s VO2max, we used the criteria described by Tanaka et al. [4] Briefly, after a 2-min warm up at 15 Watt (W), the subjects began the actual exercise protocol at a 30 W level. The workload was increased every minute by 15 W until volitional exhaustion. We determined the highest oxygen uptake achieved over 30 seconds as the VO2max.
To determine each subject’s exercise intensity during the exercise session, we used the VO2max measurement data (i.e., values per minute for workload and VO2) and calculated a simple linear regression equation for each subject: Y = β x + c, with Y = workload (W), x = VO2 (ml), and ß and c as constants. Subsequently, percentage VO2max data (ml) (e.g., 120% VO2max for SIT) were applied to the equation, whereupon each subject’s exercise intensity (workload (W)) was determined.
To determine net exercise VO2 and EPOC, the area under the curve (AUC) for each phase was calculated for each subject using a statistical-software package (SAS version 9.2, SAS Institute Inc, Cary, NC, USA). As for the SIT and HIAT, periods of active rest were included in the calculation of the net exercise VO2.
To analyze differences among the three sessions, we used one-way analysis of variance (ANOVA), and applied Tukey-Kramer’s post hoc test when the difference was significant (P < 0.05) according to the results of ANOVA. We assessed the relationship between two measurement values with Pearson’s product moment correlation. We used SAS, version 9.2 (SAS Institute Inc, Cary, NC, USA) to analyze the data.