Respiratory gas collection and analysis with indirect calorimetry allows assessment of energy and substrate use during rest and exercise and aerobic capacity. We have developed a novel system for simultaneous multiple-subject measurement of respiratory gas exchange during rest and exercise for up to five subjects, and validated the system with a series of experiments. Thirteen healthy young men (mean age 22 ± 2 years, height 172 ± 6 cm, weight 67.1 ± 11.6 kg) participated in this study. Memory problems of the mass spectrometer, which are caused by switching analyzed gases of different compositions between channels, was sufficiently eliminated with a washout of 2 s (seconds) with errors of less than 0.3% for O
2 and less than 0.4% for CO
2. The high-response mass spectrometer and specially designed mixing chamber enabled correct estimation of metabolic measurements, even for gas concentration data from a non-continuous flow of any given 1s of every 10-s interval (spot-sampling method). Diet-induced thermogenesis (DIT) and oxygen consumption (VO
2) during maximal incremental exercise were not significantly different between the novel and Douglas bag methods (
P = 0.61 and
P = 0.56, respectively). The limit of agreement of DIT and VO
2 during maximal incremental exercise between the two methods were 2.8 ± 43.6% and 0.9 ± 4.8%, respectively. We concluded that the newly developed system has reasonable validity over a wide range.
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