Aims: How endurance training alters muscle lipid metabolism while preserving insulin sensitivity remains unclear. Because acute free fatty acid (FFA) elevation by lipid infusion reduces insulin sensitivity, we hypothesized that training status would alter accumulation of muscle triacylglycerol (TAG), diacylglycerol (DAG), ceramide, and acylcarnitine during acute FFA elevation. Methods: Trained (n=15) and sedentary (n=13) participants matched for age, sex, and BMI received either a 6-hour infusion of lipid (20% Intralipid at 90 ml/h) or glycerol (2.25 g/100 ml at 90 ml/h) during a hyperinsulinemic-euglycemic clamp. Muscle biopsies were taken at 0, 120, and 360 min after infusion initiation to measure intramyocellular concentrations of TAG, DAG, ceramides, and acylcarnitines by liquid chromatography/tandem mass spectrometry. Results: Trained participants had a higher VO2 max and insulin sensitivity than sedentary participants. The lipid infusion produced comparable elevation of FFA [594 ± 90 (SE) µmol/L in trained, 721 ± 30 (SE) µmol/L in sedentary, p = 0.4] and decline in insulin sensitivity (-44.7% trained vs. -47.2% sedentary, p=0.89). In both groups, lipid infusion increased the linoleic and linolenic acid content of TAG without changing total TAG. In the sedentary group, lipid infusion increased total, oleic, linoleic acid and linolenic acid content of DAG. Regardless of training status, lipid infusion did not alter total ceramide, saturated ceramide, palmitoyl-carnitine or oleoyl-carnitine. Conclusion: During acute FFA elevation, trained adults have a similar decline in insulin sensitivity with less accumulation of muscle DAG than sedentary adults, suggesting that lipid-induced insulin resistance can occur without elevation of total muscle DAG.