Sedentary individuals combine a low capacity to oxidise intramuscular triglycerides (IMTG) with the accumulation of fatty acid metabolites that contribute to skeletal muscle insulin resistance. Trained individuals however accumulate large IMTG stores but remain insulin sensitive. The mechanism that allows trained individuals to oxidise IMTG during exercise is not fully understood. Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) control skeletal muscle lipolysis. ATGL is present on the surface of lipid droplets (LD) containing intramuscular triglyceride (IMTG) in both the basal state and during exercise. HSL translocates to LD in ex vivo electrically stimulated rat skeletal muscle. Perilipin-2 & Perilipin-5 associated lipid droplets (PLIN2+ LD & PLIN5+ LD) are preferentially depleted during exercise in humans indicating these PLINs may control muscle lipolysis. The aim of this study was to test the hypothesis that in human skeletal muscle in vivo HSL (but not ATGL) translocates to PLIN2+ LD and PLIN5+ LD during moderate-intensity exercise. Percutaneous biopsies from the m. vastus lateralis of 8 lean trained men (age 21 ± 1 years, BMI 22.6 ± 1.2 kg.m-2 and Vo2peak 48.2 ± 5.0 ml.min-1.kg-1) were obtained before and immediately following 60 min of cycling exercise at ~59% Vo2peak. Cryosections (5µm) were stained using antibodies targeting ATGL, HSL, PLIN2 and PLIN5. LD were stained using BODIPY 493/503. Images were obtained using confocal immunofluorescence microscopy and object based colocalisation analyses were performed. Following exercise the fraction of LD that colocalised with HSL increased (P < 0.05). This increase was significantly larger for PLIN5+ LD (+53%) than for PLIN5- LD (+34%) (P < 0.05), while the increases in HSL association to PLIN2+ LD (+16%) and PLIN2- LD (+28%) were not significantly different. The association of ATGL did not change for any of the LD subclasses. This study presents the first evidence of exercise-induced HSL translocation to LD in human skeletal muscle and identifies PLIN5 as a facilitator of this mechanism. Future research should aim to identify when dysregulation of this mechanism occurs in the sedentary or ageing population in the prognosis of insulin resistance.