Context:Animal studies revealed that medium-chain fatty acids (MCFA), due to their metabolic characteristics, are not stored in skeletal muscle and may therefore not give rise to potentially hazardous lipid species impeding insulin signaling.Objective:We here hypothesized that infusion of medium-chain triacylglycerols (MCT) in healthy lean subjects does not lead to ectopic fat accumulation and hence does not result in lipid-induced insulin resistance.Design and Methods:Nine healthy lean male subjects underwent a 6-h hyperinsulinemic-euglycemic clamp with simultaneous infusion of 1) a 100% long-chain triacylglycerols (LCT) emulsion, 2) a 50/50% MCT/LCT emulsion, or 3) glycerol in a randomized crossover design. Muscle biopsies were taken before and after each clamp.Results:MCT/LCT infusion raised plasma free fatty acid levels to a similar level compared with LCT infusion alone. Despite elevated free fatty acid levels, intramyocellular triacylglycerol (IMTG) levels were not affected by the MCT/LCT emulsion, whereas LCT infusion resulted in an approximately 1.6-fold increase in IMTG. These differences in muscle fat accumulation did not result in significant differences in lipid-induced insulin resistance between LCT (-28%, P = 0.003) and MCT/LCT (-20%, P < 0.001). Total skeletal muscle ceramide content as well as lactosyl- and glucosylceramide levels were not affected by any of the interventions. In addition, the distribution pattern of all ceramide species remained unaltered.Conclusions:Although we confirm that MCFA do not lead to ceramide and IMTG accumulation in skeletal muscle tissue in humans, they do induce insulin resistance. These results indicate that, in humans, MCFA may not be beneficial in preventing peripheral insulin resistance.