PROJECT ABSTRACT Cardiovascular disease (CVD) is a significant public health burden and remains the leading cause of mortality among the U.S population. CVD progression can originate during childhood, and youth with overweight and obesity (OW/OB) have an increased risk for CVD development. One mechanism that contributes to the development of CVD is imbalanced cardiac autonomic nervous system (ANS) function. The cardiac ANS governs the heart’s response to everyday conditions via sympathetic and parasympathetic modulation, in which balance of the sympathetic and parasympathetic systems indicates healthy cardiac ANS function. However, youth with OW/OB present overactivation of the sympathetic modulation of the heart, which is strongly associated with several adverse health outcomes. Studies in youth demonstrated that increased sedentary time (ST) is related to higher CVD risk. However, it is unknown whether the associations between ST and CVD are due to time displaced from physical activity to ST or due to underlying physiological processes stimulated by ST. Reduced cardiac ANS balance may be mediating the ST and CVD risk association, but few researchers have examined the associations between ST and cardiac ANS function, and none have examined associations within a day or within a person, and have not experimentally manipulated prolonged ST. Understanding the relationship of ST to cardiac ANS function may provide insight toward methodologies to alleviate CVD development, especially in youth with OW/OB who are at significantly higher risk for CVD. This project aims to leverage data collection opportunities from the NIH-funded Sedentary Breaks Study 3 (P.I. Dr. Britni Belcher; R01DK12333) to elucidate observational and experimental relationships between ST and cardiac ANS function via measures of heart rate variability in youth with OW/OB. The first phase of this project will investigate daily associations between objectively-measured ST and cardiac ANS function under habitual conditions. In the second phase, this project will have an innovative experimental component to determine the acute effects of interrupting ST on cardiac ANS function in-lab over seven consecutive days among three conditions: 1) continuous sitting (SIT), 2) sitting interrupted by 3-minute bouts of moderate-intensity walking every 30 minutes (SIT+WALK), and 3) sitting interrupted by a single 18-minute bout of moderate-intensity walking (EX).