Aerobic exercise improves skeletal muscle mitochondrial function and completeness of fatty acid beta oxidation, which contribute to reductions in lipid accumulation and improved insulin sensitivity. The aim of this study was to determine the acute effects of exercise on the completeness of beta oxidation in lean vs. overweight and obese (Ov/Ob) skeletal muscle. 30 men, aged 19–30 years, were recruited and divided into two groups: lean (BMI<25, 18.5–24.1 kg/m2, n=15) and Ov/Ob (BMI≥25, 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), participants performed an acute bout of cycling exercise (50% VO2max, spending ~650 kcal). Vastus lateralis muscle biopsy samples were collected prior to (Pre) and immediately following (Post) the acute exercise bout. In skeletal muscle, metabolomics of acylcarnitines was determined by tandem MS and data were analyzed using JMP. Pairwise correlation maps were generated for all skeletal muscle acylcarnitines, Pre and Post, in lean and Ov/Ob. Strong (correlation coefficient greater than 0.6) positive correlations were observed between long and medium chain acylcarnitine species in lean Pre and between long and short chain species in lean Post. In Ov/Ob, strong positive correlations were observed among long chain species Pre and between long, medium and short chain species Post. Ov/Ob Pre exhibited a smaller number of positive correlations between long and medium/short chain acylcarnitines compared to lean Pre, indicating more incomplete beta oxidation in Ov/Ob Pre compared to lean Pre. Ov/Ob Post exhibited a higher number of positive correlations between long and medium/short chain fatty acids compared to lean Post, indicating a shift Post exercise to more complete beta oxidation in Ov/Ob Post than in lean Post. Prior to exercise, C2, C10‐OH, C12‐OH, C18:1, C18:2‐OH, C22:4, C22:3 were increased in Ov/Ob compared to lean. Acute exercise resulted in significantly increased levels of short chain acylcarnitnes C2, C3, C4, C5:1, C5, C4‐OH and free carnitine in lean and C2 and C4‐OH in Ov/Ob, with other short chain acylcarnitines tending to increase in Ov/Ob. Medium chain acylcarnitines, C6, C8:1, C7‐DC, C10 and C8‐DC increased in lean Post compared to lean Pre; whereas, C8:1, C7‐DC, C10 and C12 increased in Ob/Ov Post compared to lean Post. There no changes in long chain acylcarnitine levels in lean Post vs. lean Pre; however, C14, C18:1, C20:2, C20:1, C20, C22:3, C22:2 and C22:1 increased in Ov/Ob Post compared to Ov/Ob Pre. Thus, exercise may increase skeletal muscle mitochondrial beta oxidation of fatty acids in both lean and Ov/Ob and may be more effective in increasing completeness of beta oxidation in Ov/Ob.Support or Funding InformationThis research was funded by a Ralph W. and Grace M. Showalter Research Trust grant and Indiana CTSI funds to TMH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.