Background: Almost 1 in 2 adults in the US has obesity, with women facing the highest prevalence of severe obesity. Emerging evidence shows that the gut microbiome and its metabolites play a key role in metabolic regulation, acting as signals in active metabolic tissues such as adipose and skeletal muscle, thereby contributing to the deterioration of cardiometabolic health in individuals with obesity. Recent findings suggest that functional characteristics of the gut microbiome, rather than compositional changes alone, may partially explain why some individuals experience greater metabolic benefits from exercise than others. We designed a pilot trial to assess the acute response to an exercise bout across distinct obesity phenotypes and to identify microbial signatures associated with lifestyle interventions. Methods: We proposed a pilot trial in which 40 young adults (21 to 40 years old) with distinct exercise (< 150 minutes per week or > 4 hours per week) and body compositions (body mass index [BMI] 18.5 to 24.99 or > 30 kg/m2) would undergo an acute exercise bout. The outcomes of this pilot trial are as follows: (1) Examine the effects of a 30-minute bout of moderate-intensity aerobic exercise (60 to 70% heart rate reserve) on the abundance and functional activity of short-chain fatty acid (SCFA)-producing gut bacteria across different obesity phenotypes in women; (2) Assess the acute effects of the same exercise bout on SCFA concentrations in stool and circulating plasma metabolomic profiles. Discussion: The results of this pilot study will inform the feasibility of a larger trial to establish the gut-synthesized