PROJECT SUMMARY/ABSTRACT Despite the well-known health benefits of exercise, most adults do not meet physical activity (PA) recommendations, and the prevalence of metabolic diseases continues to rise. Traditionally, PA is typically prescribed as a single-continuous bout of 30-60 min per day. The newly released PA guidelines no longer require PA to be in 10-min bouts and suggest reducing sedentary behaviors (SB). This is important because epidemiologic and experimental evidence suggest that breaking up SB (i.e. sitting) with short-frequent bouts of PA acutely decreased glycemia, even in individuals who exercise regularly. However, whether the acute metabolic effects of interrupting prolonged sitting are sustained over time and outside the laboratory and potential underlying biological mechanisms remain poorly understood. My dissertation aims to address these questions in sedentary, physically inactive adults with overweight to obesity. The preliminary data from my dissertation suggest that breaking up SB with short-frequent PA bouts spread throughout the day acutely increases the reliance on carbohydrate as fuel over 24h and following a meal, which may explain the decrease in daily glycemia observed in numerous studies. The objective of this F31 award is to investigate the sustained (4-week) effect of breaking up SB with short-frequent bouts of PA in overweight to obese, sedentary, physically inactive adults on glycemic control and potential underlying mechanisms (i.e. 24h and postprandial dietary and total carbohydrate oxidation), and to compare the effects induced by an intervention matched for total active time but consisting of a daily-single continuous bout of PA. The benefits of the PA spread throughout the day on glycemia compared to the daily performance of a single-continuous bout of PA is likely not due to differences in whole-body insulin sensitivity but to a greater use of carbohydrate as fuel. During the short bouts of activity, carbohydrate from glycogen stores is likely the primary energy source and then replenished by increasing skeletal muscle uptake of plasma glucose. Over time, this effect is lasting several hours after the last bout of activity which decreases postprandial glycemia and daily glucose excursions, along with greater oxidative rates of total and dietary carbohydrate even when individuals are resting. The proposed studies will provide an initial evidence base for the health benefits of breaking up prolonged sitting with short bouts of activity, and key information for future mechanistic studies. This F31 award further outlines a career development plan to supplement my doctoral training focused on the metabolic effects of PA to facilitate clinical research training in the use of stable isotope tracer methodology to track substrate metabolism, doubly labelled water method to derive total daily energy expenditure, use of accelerometry-based monitors to assess the daily patterns of SB and PA and continuous glucose moni...