The purpose of this study was to determine: 1) the reliability of 24-h respiratory calorimetry measurements, and 2) the effects of low- versus high-intensity exercise on energy expenditure (EE) and substrate oxidation over a 24-h period. Eight women (age 28 ± 4.3 yr) were measured for body composition, maximal oxygen consumption while cycling, and EE in three, 24-h calorimeter tests, with identical work output but differing intensity during a 60-min exercise session. Low-intensity (LI) exercise involved continuous cycling at 50% ˙VO2max; whereas high-intensity (HI) exercise involved interval cycling (2 min exercise/recovery) at 100%˙VO2max. Subjects were randomly assigned to the first two tests at LI or HI, with the third test at the alternate intensity. No differences in EE or respiratory quotient (RQ) during rest, sleep, exercise, or over the 24 h were found between the first two tests (C.V. = 6.0%), demonstrating the reliability of the measurements. The HI protocol elicited significantly higher EE than LI during rest, exercise, and over 24 h, whereas sleeping EE approached significance. No significant differences in RQ during rest, sleep, or over 24 h were found, but 24-h lipid and carbohydrate oxidation were similar in the two protocols. The HI exercise RQ was significantly higher than LI. These findings demonstrated higher 24-h EE in the HI than LI protocol, but similar 24-h substrate oxidation rates.