High respiratory quotient (RQ) has been associated with fat mass (FM) gain in some, but not all studies. Variability among results may reflect differences in the RQ variable measured (fasting vs. 24-h) or may be due to differences in control for factors that affect RQ, such as diet, energy balance, circulating insulin, and insulin sensitivity. The objective of this study was to determine whether different RQ values (fasting, sleeping, nonsleeping, and 24-h) would predict change in FM over 2 years in obesity-prone women, controlling for diet and adjusting for energy balance, circulating insulin, and insulin sensitivity. Participants were 33 previously overweight premenopausal women. Fasting, sleeping, nonsleeping, and 24-h RQ values were measured during controlled-diet conditions by respiratory chamber calorimetry. Intravenous glucose tolerance tests were also performed to adjust for fasting insulin, acute insulin response to glucose, and insulin sensitivity. Over the following 2 years, changes in FM were tracked annually by dual energy X-ray absorptiometry. High nonsleeping RQ (NSRQ) predicted 2-year change in FM independently of energy balance, circulating insulin, and insulin sensitivity. This observation suggests that low postprandial fat oxidation may uniquely predispose obesity-prone individuals to accrual of adipose tissue. © 2010 The Obesity Society.