The purpose of this study was to examine the rates of substrate oxidation in lean and obese women during short-duration, high-intensity exercise and to examine the effects of a 16-week exercise training program on substrate oxidation during 30 min of exercise in lean and obese individuals. Fat and carbohydrate oxidation were measured in 8 non-obese (Non-Ob), 11 lower-body obese (LBO) and 12 upper-body obese (UBO) women at rest and during 30 min of treadmill exercise at 70% of peak oxygen uptake. The obese women participated in 16 weeks of aerobic training (3 times per week at 70% of maximum oxygen uptake). Total fat and carbohydrate oxidation were measured using indirect calorimetry. The respiratory exchange ratio (R) was similar between groups at rest and was found to decrease throughout the exercise session (P< 0.01). Fat oxidation was greater at 15 min of exercise than at rest (P<0.01) but did not increase significantly more at 30 min of exercise. Obese women had significantly greater fat oxidation (both absolute concentrations and when expressed per kg of fat free mass, FFM) at 30 min of exercise than the Non-Ob women [Non-Ob 23.5 (3.7) micromol.kg FFM(-1).min(-1), LBO 35.2 (3.1) micromol.kg FFM(-1).min(-1), UBO 33.2 (2.6) micromol.kg FFM(-1).min(-1); P<0.01]. Carbohydrate oxidation also increased (P < 0.01) in response to exercise, but no group differences were found. The pattern of fat distribution (LBO vs UBO) did not affect the resting or exercise fat oxidation (P=NS). Sixteen weeks of aerobic exercise did not result in significant changes in resting or exercise fat oxidation in the obese women (n = 10; P=NS), but did significantly increase carbohydrate oxidation [pretraining 8.6 (1.4) micromol.kg FFM(-1), post-training 13.6 (2.1) micromol.kg FFM(-1).min(-1); P<0.01]. Unlike earlier studies, this shorter-duration, higher-intensity exercise resulted in a greater whole-body fat oxidation in the obese women than in the Non-Ob women, and exercise training did not result in any changes in fat oxidation, but did increase exercise carbohydrate oxidation.