Key pointsr Heavy-intensity exercise causes a progressive increase in energy demand that contributes to exercise limitation.r This inefficiency arises within the locomotor muscles and is thought to be due to an increase in the ATP cost of power production; however, the responsible mechanism is unresolved.r We measured whole-body O 2 uptake and skeletal muscle ATP turnover by combined pulmonary gas exchange and magnetic resonance spectroscopy during moderate and heavy exercise in humans.r Muscle ATP synthesis rate increased throughout constant-power heavy exercise, but this increase was unrelated to the progression of whole-body inefficiency.r Our data indicate that the increased ATP requirement is not the sole cause of inefficiency during heavy exercise, and other mechanisms, such as increased O 2 cost of ATP resynthesis, may contribute.Abstract During constant-power high-intensity exercise, the expected increase in oxygen uptake (V O 2 ) is supplemented by aV O 2 slow component (V O 2 sc ), reflecting reduced work efficiency, predominantly within the locomotor muscles. The intracellular source of inefficiency is postulated to be an increase in the ATP cost of power production (an increase in P/W). To test this hypothesis, we measured intramuscular ATP turnover with 31 P magnetic resonance spectroscopy (MRS) and whole-bodyV O 2 during moderate (MOD) and heavy (HVY) bilateral knee-extension exercise in healthy participants (n = 14). Unlocalized 31 P spectra were collected from the quadriceps throughout using a dual-tuned ( 1 H and 31 P) surface coil with a simple pulse-and-acquire sequence. Total ATP turnover rate (ATP tot ) was estimated at exercise cessation from direct measurements of the dynamics of phosphocreatine (PCr) and proton handling. Between 3 and 8 min during MOD, there was no discernableV O 2 sc (mean ± SD, 0.06 ± 0.12 l min −1 ) or change in [PCr] (30 ± 8 vs. 32 ± 7 mM) or ATP tot (24 ± 14 vs. 17 ± 14 mM min −1 ; each P = n.s.). During HVY, theV O 2 sc was 0.37 ± 0.16 l min −1 (22 ± 8%), [PCr] decreased (19 ± 7 vs. 18 ± 7 mM, or 12 ± 15%; P < 0.05) and ATP tot increased (38 ± 16 vs. 44 ± 14 mM min −1 , or 26 ± 30%; P < 0.05) between 3 and 8 min. However, the increase in ATP tot ( ATP tot ) was not correlated with theV O 2 sc during HVY (r 2 = 0.06; P = n.s.