Increased intramyocellular triglyceride (IMTG) content is found in both insulin sensitive endurance-trained subjects and insulin resistant obese/type 2 diabetic subjects. A high turnover rate of the IMTG pool in athletes is proposed to reduce accumulation of lipotoxic intermediates interfering with insulin signaling. IMTG turnover is a composite measure of the dynamic balance between lipolysis and lipid synthesis; both are influenced by mitochondrial fat oxidation and plasma free fatty acid availability. Therefore, more attention should be given to the factors controlling the rate of turnover of IMTG. In this review particular attention has been given to muscle oxidative capacity, plasma free fatty acid availability, IMTG hydrolysis (lipolysis) and synthesis. A higher oxidative, lipolytic and lipid storage capacity in the muscle of endurance-trained subjects reflect a higher fractional turnover of the IMTG pool. Thus, the co-localization of intermyofibrillar lipid droplets and mitochondria allows for a fine coupling of lipolysis of the IMTG pool to mitochondrial -oxidation. Conversely, reduced oxidative capacity and a mismatch between IMTG lipolysis and -oxidation might be detrimental to insulin sensitivity by generating several lipotoxic intermediates in sedentary populations including obese/type 2 diabetic subjects. Further studies are clearly required to better understand the relationship between the rate of turnover of IMTG and the accumulation of lipotoxic intermediates in the pathophysiology of insulin resistance.