A pulse ([14 C] palmitate)-chase ([3 H] palmitate) approach was used to study intramuscular triglyceride (imTG) fatty acid and plasma free fatty acid (FFA) kinetics during exercise at∼ 45% peak O 2 consumption in 12 adults. Vastus lateralis muscle was biopsied before and after 90 min of bicycle exercise; 3 H 2 O production, breath 14 CO 2 excretion and lipid oxidation (indirect calorimetry) rates were measured during exercise. Results: during exercise, 8.2±1.2 and 8.4±0.7 μmol· kg− 1· min− 1 of imTG fatty acids and plasma FFA, respectively, were oxidized according to isotopic measurements. The sum of these two values was not different (P= 0.6) from lipid oxidation by indirect calorimetry (15.4±1.6 μmol· kg− 1· min− 1); the isotopic and indirect calorimetry values were correlated (r= 0.79, P< 0.005). During exercise, imTG turnover rate was 0.32±0.07%/min (6.0±2.0 μmol of imTG· kg wet muscle− 1· min− 1) and plasma FFA were incorporated into imTG at a rate of 0.7±0.1 μmol· kg wet muscle− 1· min− 1. The imTG pool size did not change during exercise. This pulse-chase, dual tracer appears to be a reasonable approach to measure oxidation and synthesis kinetics of imTG.