We have previously shown that a higher 24-h respiratory quotient (24-h RQ) predicts greater ad-libitum food intake and that nighttime eaters (NE) ingested more calories during an in-patient food intake study and gained more weight over time. We investigated whether 24-h RQ was higher in individuals who exhibited nighttime eating behavior. Healthy nondiabetic Pima Indians (PI; n = 97, 54 male/43 female) and whites (W; n = 32, 22 male/10 female) were admitted to our Clinical Research Unit. After 3 days of a weight maintaining diet, 24-h energy expenditure (24-h EE), 24-h RQ, rates of carbohydrate (CHOX) and lipid oxidation (LIPOX), and spontaneous physical activity (SPA) were measured in a metabolic chamber whereas volunteers were in energy balance and unable to consume excess calories. Individuals subsequently ate ad libitum from a computerized vending machine for 3 days with amount and timing of food intake recorded. Fifty-five individuals (36%; 39 PI, 16 W) were NE, who ate between 11 PM and 5 AM on at least one of the 3 days on the vending machines. There were no differences in BMI or percentage body fat between NE and non-NE. After adjusting for age, sex, race, fat-free mass, fat mass, and energy balance, NE had a higher 24-h RQ (P = 0.01), higher CHOX (P = 0.009), and lower LIPOX (P = 0.03) and higher 24-h SPA (P = 0.04) compared to non-NE. There were no differences in adjusted 24-h EE or sleep RQ between the groups. Individuals with nighttime eating behavior have higher 24-h RQ, higher CHOX and lower LIPOX, a phenotype associated with increased food intake and weight gain.