The aim of this study was to investigate the effects of sleep deprivation on autonomic and endocrine functions during the day and on exercise tolerance in the evening. Ten healthy young males completed two, 2-day control and sleep deprivation trials. For the control trial, participants were allowed normal sleep from 23:00 to 07:00 h. For the sleep deprivation trial, participants did not sleep for 34 h. Autonomic activity was measured from 19:00 h on day 1 to 16:00 h on day 2 by frequency-domain measures of heart rate variability. Endocrine function was examined by measuring adrenocorticotropic hormone and cortisol from venous blood samples collected on day 2 at 09:00, 13:00, and 17:00 h and immediately after an exercise tolerance testing. Autonomic regulation, particularly parasympathetic regulation estimated from the high-frequency component of heart rate variability analysis, was significantly higher in the sleep deprivation trial than in the control trial in the morning and afternoon of day 2. Plasma adrenocorticotropic hormone concentrations were significantly higher at 09:00 and 13:00 h of day 2 under sleep deprivation. Heart rate during exercise was significantly lower following sleep deprivation. Therefore, the effects of sleep deprivation on autonomic regulation depend on the time of the day.