PROJECT SUMMARY Many authorities recommend the Mediterranean diet (MedDiet) for the prevention of cardiometabolic disease. These dietary recommendations are based on population averages and may not be best suited for a given individual. Preliminary data from our group and others support that a specific dietary intervention may have highly variable effects in different individuals due to the individual composition of the gut microbiome. Furthermore, we recently reported that autologous fecal microbiota transplantation (aFMT) derived from the time of maximal weight reduction enhanced the effects of MedDiet on maintaining cardiometabolic health in an RCT. This background supports our central hypothesis that the gut microbiome can modify the effects of MedDiet on cardiometabolic disease risk. However, no studies have utilized longitudinally collected data from RCTs to test this hypothesis. Most diet-microbiome studies are limited by the use of 16S rRNA gene sequencing yielding only very general taxonomic profiling, thus omitting strain-specific diet-related biochemical functions of microbes. To gain more advanced mechanistic insights, combining shotgun metagenomics and metatranscriptomics and metabolomics in an integrated framework presents a unique opportunity to probe both the composition and functionality of gut microbial communities. This proposed project will leverage two long-term dietary RCTs, the recently completed 18-month DIRECT-PLUS trial of 294 participants and the ongoing 3-year MIND trial of 604 participants, to examine whether individual gut microbial features modify the effects of MedDiet interventions on cardiometabolic risk and body adiposity (Aim 1) and identify metabolites in feces and metabolites in plasma of gut microbial origin that explain inter-individual differences in post-intervention changes in cardiometabolic risk and adiposity (Aim 2) in the DIRECT-PLUS trial. Findings from Aims 1 and 2 will be tested for replication in the MIND trial. In Aim 3, we will investigate long-lasting, post-intervention effects of combined MedDiet and aFMT, and characterize gut microbial changes during and after the interventions in an RCT of 90 participants. Our proposal addresses a major research priority, precision nutrition, emphasized in the 2020–2030 Strategic Plan for NIH Nutrition Research and is directly responsive to PAR-19-377: “Omics-guided Biobehavioral Interventions for Improved Health Outcomes: A Step Forward in Translation” as one of the first efforts to prospectively assess the functional role of gut microbiome in explaining inter-individual heterogeneity in response to dietary interventions. Built on existing biospecimen and data collected in two well-conducted long-term RCTs and state- of-the-art multi'omics technologies, our proposal is a highly cost-efficient opportunity to generate translatable, personalized dietary interventions grounded in reproducible biological mechanisms and contribute to the paradigm shift towards p...