The largest component of daily energy expenditure is resting energy expenditure as reflected in overnight metabolic rate (OMR) and sleeping metabolic rate (SMR). Here, we determined the variation in OMR (24:00-6:00 h) and SMR values (3 h intervals) as affected by physical activity (PA) during the day and the night. Subjects were 32 females and 17 males, age 18-52 years. Energy expenditure (EE) was measured for 36 h in a whole room calorimeter (14 m3), starting in the evening, providing values before and after behavioral limitation. The mean intra-individual coefficient of variation was 1.8+/-1.4% for SMRmin (minimum EE), 2.8+/-2.0% for SMRact (minimum PA), 2.4+/-2.5% for SMRres (minimum residual EE, residual calculated from 24 h relationship between EE and PA) and 2.8+/-2.2% for OMR (n=49). Mean clock time for SMR ranged from 3:15 till 4:13 h. EE and PA increased in the hour before awakening. Surprisingly, OMR showed a significant 2.7% increase (P<0.05) during the second night of the 36 h measurement, but only for a second visit, and was related to increased physical activity during night period (R2=0.50, P<0.001). OMR measurements following unrestricted daily activity showed identical results for first and second (repeat) visits: 6.82+/-0.86 MJ/day and 6.79+/-0.93 MJ/day (n=49), respectively. It is advised to measure SMR based on minimum residual EE during nights following free-living conditions, and to exclude EE measures 1 h before awakening from SMR and OMR calculations to prevent influences of habitual wake-up time.