The interface between lipid droplets (LDs) and cellular environment is regulated by the perilipin (PLIN) proteins. In addition to many distinct roles, a common role for these proteins is inhibition of lipolysis. We investigated the effect of high fat diet and/or physical activity on three lipid droplet coat proteins (PLIN2, PLIN3 and PLIN5) and on factors regulating intramyocellular triglyceride metabolism in skeletal muscle. Insulin resistance was induced by a high-fat (HF) diet for 19 weeks in C57BL/6J mice, which were either sedentary or with access to running wheels. The abundance of fatty acid transporter FAT/CD36 together with PLIN5 was significantly greater in HF animals. We found modest changes in the protein expression of adipose triacylglycerol lipase (ATGL) and PLIN2, but no difference in glycerol-3-phosphate acyltransferase (GPAT) and PLIN3 was found. After HF feeding, the fatty acid flux into muscle may be increased via FAT/CD36, together with the augmented abundance of specific LD associating protein. The importance of the absence of significant changes in ATGL and GPAT protein abundance despite increased muscle lipid storage remains to be further elucidated.