The role of ingested fat in the etiology of obesity is controversial. The aims of this study were to determine the contributions of ingested fat oxidation to: 1) 24-h total energy expenditure (TEE), and 2) substrate oxidation during acute stationary cycle exercises in adult humans. Healthy, moderately obese (n = 18; BMI = 31 ± 1 kg/m2) subjects (8 men; 10 women) were each studied in a whole-room calorimeter for 24 h. They were fed mixed meals (55, 30, and 15% as energy from carbohydrate, fat and protein, respectively) to maintain energy balance. Each subject performed 1255-kJ cycle exercises at 50% VO 2max in the calorimeter. Study test meal fat was labeled with carbon-13 (13C). Ingested fat oxidation was estimated from breath 13CO2 excretion and the subject's chamber CO2 production. Total fat and carbohydrate oxidations were estimated from nonprotein respiratory quotient (NP-RQ) values. Endogenous fat oxidation was estimated as the difference between total fat and ingested fat oxidations. TEE was estimated from gas exchanges; 28 ± 3% of ingested fat was oxidized and it provided 8 ± 1% of 24-h TEE. During cycle exercises, ingested fat provided 50% of total fat oxidized and 13.0 ± 2% of energy expended. Endogenous fat oxidation contributed 10.4 ± 3% of energy expenditure during cycle exercises. This study extended to 24-h observations of previous studies that lasted 6-9 h on ingested fat oxidation in humans. Understanding the factors that promote ingested fat oxidation could lead to more effective obesity intervention programs. © 2005 American Society for Nutritional Sciences.