ABSTRACT Cocoa flavanols, from the seeds of theobroma cacao, the “cocoa” tree, have therapeutic properties that may improve lower extremity perfusion and reverse gastrocnemius muscle abnormalities that contribute to disability in people with lower extremity peripheral artery disease (PAD). Our funded COCOA PAD II Trial (R01-AG068458) is a multi-center double-blinded randomized trial that will test the effects of cocoa flavanols vs. placebo on change in six-minute walk distance at six month follow-up in people with PAD. Secondary outcomes include gastrocnemius perfusion, physical activity, brachial artery flow-mediated dilation and gastrocnemius muscle biopsy measures of mitochondrial activity and skeletal muscle health. Preliminary evidence supports our hypothesis that gut microbiota may mediate the beneficial effects of cocoa flavanols. First, gut microbiota metabolize cocoa flavanols to produce metabolites that enter the circulation and reach target organs where these metabolites may exert beneficial effects. Second, cocoa flavanols increase abundance of Bifidobacteria and Lactobacillus spp. but decrease Clostridium spp. in the gut microbiome. These microbiome changes have been associated with improved muscle function and exercise ability as well as reduced oxidative stress. Therefore, we request an Administrative Supplement (PA 20-227) for a 12 month period to support collection of stool and plasma samples and preparation of samples for analyses. Collected samples will be sent to Dr. Eric Pamer’s laboratory at the U. of Chicago for analyses. During this one year of funding, we anticipating collecting samples from approximately 50 participants at baseline and approximately 40 participants at six-month follow-up who are participants in the COCOA PAD II Trial. We will test the following specific aims: First, among people with PAD, we will assess whether cocoa flavanols, compared to placebo, favorably alter gut bacterial diversity, composition, and gut-microbe associated metabolomics at six-month follow-up. Second, among people with PAD randomized to cocoa flavanols, we will assess whether more favorable changes in gut bacterial diversity, composition, and gut microbe related metabolites between baseline and 6-month follow-up are associated with greater improvement in six-minute walk and other study outcomes at 6-month follow-up. Third, among people with PAD randomized to cocoa flavanols, we will assess whether baseline characteristics of the gut microbiome are associated with the degree of response to cocoa flavanols, measured by greater improvement in six-minute walk distance and other outcomes at 6-month follow-up. If our hypotheses are correct, this trial will, for the first time, establish the gut microbiome as a critical mediator of the effects of cocoa flavanols on improved walking performance in humans. Results will also delineate a key biologic pathway of cocoa flavanol therapy, and identify the gut microbiome as an important therapeuti...