Repeated bouts of acute aerobic exercise improve skeletal muscle mitochondrial function and completeness of fatty acid β‐oxidation which contributes to reductions in lipid accumulation. We hypothesize that epigenetic regulation of metabolic genes due to exercise may lead to increased mitochondrial function in overweight/obese (Ov/ob) individuals. 30 men, aged 19–30 years, were recruited and divided into two groups: lean (BMI, 18.5–24.1 kg/m2, n=15) and Ov/ob (BMI, 25.5–36.9 kg/m2, n=15). Four hours after eating a standardized high‐carbohydrate breakfast (7 kcal/kg; 60% carbohydrate, 25% fat, 15% protein), they performed an acute bout of cycling exercise (50% VO2max, spending ~650 kcal). Thirty minutes before eating the breakfast, vastus lateralis muscle biopsy samples were collected (pre). Biopsy samples were also collected immediately after (post) exercise. In skeletal muscle, metabolomics of acylcarnitines was determined by tandem mass spec and gene expression by RNA‐seq, via the Illumina platform. For analysis, TopHat was used for sequence alignment and Cuffdiff for gene expression. Mononucleosomal DNA was used in downstream MNase‐seq via the Illumina platform. In skeletal muscle, as per correlation heat maps of acylcarnitine profile, Ov/ob pre exhibited fewer positive correlations between long and medium/short chain fatty acid concentrations compared to lean pre. Exercise increased completeness of β‐oxidation of fatty acids in both lean and Ov/ob. However, Ov/ob post exhibited a higher number of positive correlations between long and medium/short chain fatty acids compared to lean post, thus indicating more complete β‐oxidation following exercise than lean. This indicates that exercise is able to increase skeletal muscle mitochondrial function in both groups but may be more beneficial in increasing mitochondrial function in Ov/ob. ~97% of all sequenced reads from RNA‐seq aligned to the genome. In our preliminary RNA‐seq results (N=4 per group), we found 62, 149 and 99 genes differentially expressed between lean pre‐Ov/ob pre, lean pre‐post and Ov/ob pre‐post, respectively. In MNase‐seq analysis, whole genome nucleosomes maps were created, which showed differential chromatin architecture dependent on overweight/obesity and on exercise training. These results show that exercise‐induced alterations in mitochondrial function in lean and Ov/ob individuals following a single, acute bout of exercise at moderate intensity are associated with epigenetic alterations and differential gene expression. Differential expression of epigenetically regulated genes may account for the increased responsiveness in mitochondrial function contributing to more complete β‐oxidation in overweight/obese individuals.Support or Funding InformationThis study was supported by funding from the Ralph W. and Grace M. Showalter Research Trust and the Indiana NIH Clinical and Translation Research Institute Core grants awarded to T.M.H.