The aim of the present study was to determine whether or not voluntary breath-holding time (BHT) changes with the time of the day. BHT with airways closed at end-expiration was measured in six male subjects in the sitting position during the morning (08.00–12.00 hours, on days 1, 6, 7 and 8) and evening (20.00–24.00 hours, on days 2 and 4). BHT increased with the number of days of testing and, at day 8, the morning values averaged 160% of those on day 1. Also, Δ P ACO 2 [the difference between end-tidal partial pressure of CO 2 ( P CO 2 ) and alveolar P CO 2 ( P ACO 2 ) at the breaking point] increased in proportion to BHT. Hence the BHT/Δ P ACO 2 ratio remained nearly constant. Voluntary hyperventilation prolonged BHT and increased Δ P ACO 2 . Conversely, in hypoxia (13% O 2 for 1–2 h), BHT and Δ P ACO 2 were reduced proportionally. During the evening sessions, most of the BHT/Δ P ACO 2 ratios in normoxia, hypoxia or after hyperventilation were higher than the corresponding morning values, with the group difference reaching statistical significance for the measurements in normoxia and hypoxia. In conclusion, voluntary BHT varies in both duration and its relationship with Δ P ACO 2 between the morning and evening hours. The results should also imply that, with an interruption of breathing, changes in alveolar and arterial gases are not the same at different times of the day.