Development of type 2 diabetes (T2D) is preceded by prediabetes, which is a metabolically "atypical" state associated with chronic low-grade inflammation, overweight and obesity, lack of exercise, and detrimental changes to the gut microbiome. Dietary intake and exercise are modifiable lifestyle factors for reducing T2D risk; however, several questions remain unanswered related to the efficacy and role of prebiotics and exercise, and their respective influences on gut microbiome composition, intestinal permeability, insulin sensitivity and metabolic flexibility. Sedentary to recreationally active overweight and obese adults 40-75 years old at-risk for T2D were recruited (n=22) and randomized to either supplementation with inulin, a prebiotic dietary fiber, (10g/d) or maltodextrin while consuming a controlled diet for six weeks. At baseline and week 6, participants completed a stool collection, a 4-sugar probe test, an intravenous glucose tolerance test (IVGTT), and high-fat meal challenge with skeletal muscle biopsies to evaluate changes in the gut microbiome composition, intestinal permeability, insulin sensitivity and metabolic flexibility, respectively. There were no baseline group differences (all p>0.05). Following the intervention, Bifidobacteria operational taxonomic units increased in the intervention group ([placebo: Δ 9.5 ± 27.2 vs inulin: 96.3 ± 35.5][p=0.03]). There were no other group differences over time in any other outcome variables with the exception of changes in metabolic flexibility. Secondarily, a systematic review of literature was conducted to determine the influence of exercise engagement on gut microbiome composition. Overall, exercise interventions appeared to diversify taxa within the Firmicutes phylum, and specifically in several taxa associated with butyrate production and gut barrier function. Due to unclear risk of bias in all studies and low quality of evidence, additional research is needed using well- designed trials. In summary, the respective influences of prebiotics and exercise on human gut microbiome composition and their subsequent effects on metabolic function and disease risk are not well understood.