In skeletal muscle, an accumulation of lipid droplets (LDs) in the subsarcolemmal space is associated with insulin resistance, but the underlying mechanism is not clear. We aimed to investigate how the size, number and location of LDs are associated with insulin sensitivity and muscle fiber types, and are regulated by aerobic training and treatment with an erythropoiesis-stimulating agent (ESA) in healthy young untrained males. LD analyses were performed by quantitative transmission electron microscopy and insulin sensitivity was assessed by a hyperinsulinemic euglycemic clamp. At baseline, we found that only the diameter (and not the number) of individual subsarcolemmal LDs was negatively associated with insulin sensitivity (R2 = 0.20, P = 0.03, n = 29). Despite 34% (P = 0.004) fewer LDs, the diameter of individual subsarcolemmal LDs was 20% (P = 0.0004) larger in type 2 fibers than in type 1 fibers. Furthermore, aerobic training decreased the size of subsarcolemmal LDs in the type 2fibers, and ESA treatment lowered the number of both intermyofibrillar and subsarcolemmal LDs in the type 1fibers. In conclusion, the size of individual subsarcolemmal LDs may be involved in the mechanism by which LDs are associated with insulin resistance in skeletal muscle.