Background: Fat balance is primarily dependent upon the state of energy balance1. There is furthermore a strong positive association between daily activity energy expenditure and 24hr dietary fat oxidation2. The respective role of the terms of exercise (energy expenditure, intensity, duration and frequency) on nutrient metabolism is still poorly known. Purpose: To compare the metabolic effects of 4 days of activity microbursts (MICRO: 5 minutes of moderate intensity walking every hour for 9 hours) to an isocaloric single 45-min bout of moderate intensity walking (ONE) and a sedentary control condition (SED) in overweight adults. Methods: In this ongoing study, eleven (8F/3M, mean±SD: age=32±7 yrs, BMI=31.0±1.9 kg/m2) subjects were studied under 3 different conditions (MICRO, ONE, SED) in random order. Each condition consisted of 3 days in a free living state followed by a 24hr stay in a whole room calorimeter to measure total energy expenditure (TEE) and substrate utilization. Energy intake was controlled and matched across days and conditions by design. Protein oxidation was estimated by urinary nitrogen excretion. The breakfast meal contained a fatty acid stable isotope (1-13C oleic acid, 20mg/kg mixed in breakfast), and 24 h dietary fat oxidation was quantified based on exhaled 13CO2 in breath samples. Results: As expected, 24hr TEE increased in both active conditions and resulted in negative energy balance (ONE: -381±140 kcal; MICRO: -414±125 kcal) compared to SED (-29±136 kcal, p<0.05 for both). Compared to the SED condition, 24hr total fat oxidation increased in ONE (69.4±26.6 g/d vs. 92.1±30.9 g/d respectively, p=0.048), but 24hr carbohydrate oxidation increased in MICRO (299.3±70.6 g/d vs. 364.9±82.3 g/d respectively, p=0.016). Protein oxidation did not differ between conditions. 24hr dietary fat oxidation tended to be greater in ONE compared to SED (36.2±12.3 vs. 30.8±4.8 % dose recovery respectively, p=0.08), but not in MICRO (31.3±6.8 % dose recovery, p>0.05). There was a strong correlation between changes in 24hr dietary fat oxidation induced by one continuous bout of exercise and changes in 24hr TEE (r2=0.68, p=0.003), but no relationship existed when the increase in 24hr TEE was induced by microbouts of exercise (r2=0.014, p=0.7). Conclusion: While both active conditions increase energy expenditure and create an energy deficit to a similar extent, they had different effects on substrate and dietary fat oxidation. Energy expenditure may not be the key factor in the regulation of lipid metabolism, and other parameters, like the frequency and/or the duration of exercise, may play a role. These preliminary results need to be confirmed once the full study is complete.