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Both Roux-en-Y gastric bypass (RYGB) surgery and exercise can improve insulin sensitivity in individuals with severe obesity. However, the impact of RYGB with or without exercise on skeletal muscle mitochondria, intramyocellular lipids, and insulin sensitivity index (S_I) is unknown. We conducted a randomized exercise trial in patients (n = 101) who underwent RYGB surgery and completed either a 6-month moderate exercise (EX) or a health education control (CON) intervention. S_I was determined by intravenous glucose tolerance test. Mitochondrial respiration and intramyocellular triglyceride, sphingolipid, and diacylglycerol content were measured in vastus lateralis biopsy specimens. We found that EX provided additional improvements in S_I and that only EX improved cardiorespiratory fitness, mitochondrial respiration and enzyme activities, and cardiolipin profile with no change in mitochondrial content. Muscle …