The number of contemporary diet plans promoting high protein intakes for weight management has increased dramatically. Complementing this dietary approach with increased physical activity has proven to be beneficial. Recent studies have suggested that protein intakes in excess of the current Recommended Dietary Allowance (0.8 g/kg) may be of metabolic benefit during weight loss. This investigation assessed changes in resting energy expenditure and substrate oxidation in overweight and obese premenopausal women in response to a weight loss intervention that combined a high-protein, reduced-calorie diet with increased physical activity. Thirty-nine overweight and obese premenopausal women (age, 30.9 +/- 1.5 years; body mass index, 30.2 +/- 0.5 kg/m2) participated in a 10-week weight loss program in which they ate a reduced-calorie diet for which protein provided 30% of total energy and approximated 1.4 g/kg. Subjects incrementally increased physical activity (ie, steps walking) throughout the diet intervention period. Resting energy expenditure, substrate oxidation, and body composition were assessed before (PRE) and after (POST) the 10-week weight loss program. Subjects experienced a 5% decrease in body weight, with significant decreases in both fat mass (PRE, 35.5 +/- 1.2 kg; POST, 32.4 +/- 1.1 kg; P < .0001) and fat-free mass (PRE, 44.6 +/- 0.7 kg; POST, 43.6 +/- 0.7 kg; P < .0001). Changes in body weight or body composition did not alter resting energy expenditure. Protein oxidation increased (PRE, 18% +/- 1%; POST, 20% +/- 1%; P < .05) and fat oxidation decreased (PRE, 37% +/- 3%; POST, 30% +/- 3%; P < .05) after the 10-week intervention. These findings illustrate that a weight loss intervention combining consumption of a high-protein, reduced-calorie diet with increased physical activity promotes weight loss without negatively impacting resting energy expenditure in this population of women.