OBJECTIVE: Determination of within-subject day-to-day coefficients of variations (CV) of 24-h measurements of spontaneous physical activity (SPA), macronutrient oxidation, energy expenditure (EE) and heart rate (HR). DESIGN: Two 24-h measurements in respiration chamber, separated by 14 days of strict weight stability. SUBJECTS: 22 healthy obese subjects, 15 females and seven males (age 27 - 64 y, BMI: 30 - 39 kg/m²). MEASUREMENTS: SPA assessed by microwave radar, substrate oxidation and EE measured by indirect calorimetry and heart rate measured by telemetry. RESULTS: For 24-h measurements CV was 0.8% for RQ, 6.7% for fat oxidation, 5.6% for carbohydrate oxidation, 14.5% for protein oxidation, and 4.3% for heart rate. SPA assessed by microwave radar was also reproducible (CV = 9.7%). Within-subject day-to-day coefficients of variations (CV) were 1.5 and 1.8% for 24-h EE and sleeping EE, and 4.1% for RMR. Both on day 1 and 14, 93.4% of the variation in 24-h EE could be explained by differences in fat-free mass (92.3 and 92.4%, respectively) and SPA (1.1 and 1.0%, respectively). Also the algorithm and correlation between 24-h RQ and 24-h energy balance were highly reproducible (r = 0.56, P < 0.01 and r = 0.48, P < 0.05). CONCLUSION: Both macronutrient oxidation, SPA and EE in obese patients can be determined with high reprodicubility in a respiration center.