Hormone sensitive lipase (HSL) catalyzes the hydrolysis of intramuscular triacylglycerol (IMTG), however its regulation in skeletal muscle is poorly understood. To examine the effects of reduced free fatty acid (FFA) availability on HSL activity in skeletal muscle during aerobic exercise, 11 trained males exercised at 55% VO₂max for 40 min following the ingestion of nicotinic acid (NA) or nothing (Control). Muscle biopsies were taken at rest and 5, 20, and 40 min of exercise. Plasma FFA were suppressed (P<0.05) in NA during exercise (0.49 ± 0.10 vs. 0.11 ± 0.01 mM). The respiratory exchange ratio (RER) was increased throughout exercise (0.020 ± 0.008) following NA ingestion. However, the provision of energy from fat oxidation only decreased from 33% of the total in the Control trial to 26% in the NA trial, suggesting increased IMTG oxidation in the NA trial. Mean HSL activity was 2.25 ± 0.15 mmol/kg dm/min at rest and increased (P<0.05) to 2.94 ± 0.20 mmol/kg dm/min at 5 min in Control. Contrary to the hypothesis, mean HSL was not activated to a greater extent in the NA trial during exercise (2.20 ± 0.28 at rest to 2.88 ± 0.21 mmol/kg dm/min at 5 min). No further HSL increases were observed at 20 or 40 min in both trials. There was variability in the response to NA ingestion, as some subjects experienced a large increase in RER and decrease in fat oxidation, while other subjects experienced no shift in RER and maintained fat oxidation despite the reduced FFA availability in the NA trial. However, even in these subjects, HSL activity was not further increased during the NA trial. In conclusion, reduced plasma FFA availability accompanied by increased epinephrine concentration did not further activate HSL beyond exercise alone.