SUMMARY Exercise is an extremely effective lifestyle intervention that dramatically lowers the risk for cardiovascular, metabolic, neoplastic, chronic inflammatory, and neurodegenerative diseases. Despite these beneficial effects, the modern human lifestyle is highly sedentary and new approaches to understanding and improving exercise performance are urgently needed. In this proposal, we will take a new approach and investigate physical activity through the lens of the gastrointestinal tract. In preliminary work using gnotobiotic mice and microbiota depletion approaches, we have recently discovered a critical role for the intestinal microbiome in regulating exercise performance. We found that intestinal microbial colonization contributes to the exercise-induced surge in dopamine in the brain. Dopamine, in turn, is a major element of the reward and reinforcement centers of the brain that drive the engagement in physical activity. Importantly, elevating dopamine levels in the striatum of microbiota-depleted mice restores their exercise performance. This effect of the microbiota on the brain during exercise is dependent on TRPV1+ afferent sensory neurons. These findings provoke the central hypothesis that that the microbiome effect on exercise performance is mediated by neuronal gut-brain signaling which regulates the availability of dopamine in the striatum. We will employ an innovative toolbox at the interface of microbiome science, exercise physiology, and neuroscience to address three central questions: (1) Which microbial genes and which intestinal metabolites influence exercise performance? (2) Which dopamine-sensitive neurons respond to exercise and the microbiome to enhance physical activity? (3) Which sensory neurons transmit the microbiome-derived gastrointestinal signal to the brain to enhance exercise performance? Collectively, these studies will mechanistically define elements of a gut-brain pathway linking the commensal microbiota, gut-innervating sensory neurons, and striatal activity during exercise. Further, the proposed experiments will provide important pre-clinical evidence on the therapeutic potential of gastrointestinal interventions, such as diet and microbial modulation, aimed at enhancing physical activity.