Although public-health recommendations to engage in moderate-to-vigorous physical activity (> 30min, 5 d/wk) have been widely promulgated by the governments, most people in contemporary societies are physically inactive. Sedentary behaviors, like time spent sitting, are associated with serious implications on metabolic health, indicating that even in individuals who regularly exercise a reduction in time spent sitting can confer health benefits1. Metabolic flexibility, defined as the ability to adjust substrate use to substrate availability and energy demand, has been recognized as a core component of metabolic health. This study tests the effect of interruptions of prolonged sitting by microbursts of activity on metabolic flexibility. We are comparing the metabolic effects of 4 days of activity microbursts (5min of moderate intensity walking every hour for 9h) to an isocaloric single 45-min bout of moderate intensity walking and a sedentary control condition in overweight adults. In this ongoing study, eleven (age=32±7 yrs, BMI=31.0±1.9 kg/m2) subjects out of 24 were studied under 3 different conditions in random order. Each condition consisted of 3 days in a free living state followed by a 24h stay in a whole room calorimeter to measure total energy expenditure and substrate utilization. Energy intake was controlled and matched across days and conditions by design. Protein oxidation was estimated by urinary nitrogen excretion. Blood samples were collected every hour from 8am to 10pm, at 3am and the following day at 7am to measure plasma insulin, glucose and free fatty acids. Metabolic flexibility is assessed by the daily changes in plasma insulin, glucose and free fatty acids concentration and non protein respiratory quotient (NPRQ). As previously published2, a metabolic inflexible state is characterized by a large daily variance in insulin for a small variance in NPRQ, i.e. a small shift in fuel mix being oxidized at a high insulin signal. We expect the metabolic effects of microbursts of activity to be mediated through enhanced insulin sensitivity and a greater shift in substrate use in response to meal consumption. Plasma samples are under analysis. We believe the results from this study will provide an initial evidence base for the health benefits of breaking up prolonged sitting with short bursts of activity. A finding that short bouts of activity promote metabolic flexibility will be of importance for individuals who have difficulty complying with traditional physical activity recommendations that promote structured exercise to be done in one longer bout.