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Summary: The tissue-specific release and uptake of metabolites in response to exercise is incompletely understood. Here, we detail a protocol to assess arteriovenous differences across the liver and hindlimb muscles in response to treadmill exercise in mice. We describe steps for the treadmill running of mice and the region-specific sampling of blood from the liver and hindlimb. This procedure is particularly relevant for the study of tissue-specific metabolism in response to exercise.For complete details on the use and execution of this protocol, please refer to Sato et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.