PROJECT SUMMARY & ABSTRACT Does time of day of exercise and nutrient status prior to exercise affect the 24-hour (h) metabolic response? The objective of this proposal is to determine the effect of aerobic exercise performed in the morning (AM) or evening (PM) under fasted and fed conditions on 24h energy expenditure (EE), substrate oxidation, hormonal profiles, and key metabolites in adults with overweight and obesity. Nearly two-thirds of US adults who attempt to lose weight engage in exercise as a primary strategy. However, weight loss from exercise alone is highly variable and less than predicted. Thus, strategies to enhance weight and fat loss from aerobic exercise are desperately needed. Two promising strategies that may help to promote fat loss are exercising in the morning and exercising while fasted. Our preliminary data, as well as others’ data, suggest that morning exercise promotes greater fat loss, and this enhanced fat loss may be due to increased EE and fat oxidation. Further, engaging in aerobic exercise in the post-absorptive state (fasted) has been shown to increase fat oxidation, free fatty acid mobilization, transient energy deficit, and β-hydroxybutyrate concentration compared to post-prandial (fed) exercise. These clinical data are supported by several recent preclinical studies delineating the energetic responses to AM vs. PM exercise in rodent models. Importantly, AM exercise favors fatty acid and amino acid metabolism while PM exercise enriches glycolytic pathways. One limitation of these preclinical investigations is that nutrient status prior to exercise (fasted vs. fed) was not controlled and evaluated. Nutrient status affects subsequent energy metabolism with fasted exercise promoting glycogen depletion and fatty acid mobilization. Therefore, fasted AM exercise may be the optimal treatment paradigm for promoting fat loss. However, a rigorously controlled study examining the effect of both exercise timing and nutrient status during exercise on 24h energy metabolism is needed. In the proposed study, adults with overweight or obesity will complete a baseline, no-exercise control visit, followed by 4 exercise conditions (Fasted-AM, Fed-AM, Fasted-PM, Fed- PM) in a randomized order, separated by a 2-5 week washout period. Each condition will include an acclimation period, eucaloric and macronutrient-controlled diet, and control of physical activity, sleep, and meal timing. This study will measure 24h components of EE and substrate oxidation using whole-room indirect calorimetry (Aim 1) and untargeted and targeted plasma metabolites and hormonal/energy profiles (Aim 2). Plasma metabolites and hormonal/energy profiles will be examined using serial blood draws immediately following exercise every 1- 2h across the 24h in-patient visit. The central hypothesis is that fasted AM exercise will result in the most robust metabolic response that is optimal for fat and weight loss followed by Fasted-PM, Fed-AM, Fed-PM, respectively. T...