(-)-Hydroxycitrate (HCA), a competitive inhibitor of ATP-citrate lyase, should reduce the extramitochondrial acetyl-CoA pool. It has been hypothesized that HCA ingestion can reduce malonyl-CoA concentrations and consequently increase fatty acid oxidation in vivo.This study investigated the acute effects of HCA supplementation on substrate utilization at rest and during exercise in endurance-trained humans.Ten cyclists [x+/- SD) age: 24 +/- 2 y, weight: 73 +/- 2 kg, maximal oxygen uptake: 4.95 +/- 0.11 L/min, maximal work output (W:max): 408 +/- 8 W] were studied at rest and during 2 h of exercise at 50% W:max on 2 occasions. Both 45 and 15 min before exercise and 30 and 60 min after the start of exercise, 3.1 mL/kg body wt of an HCA solution (19 g/L) or placebo was ingested. Total fat and carbohydrate oxidation rates were assessed. Blood samples were collected at 15-min intervals at rest and every 30 min during exercise.Plasma HCA concentrations increased after HCA ingestion up to 0.39 +/- 0.02 mmol/L (82.0 +/- 4.8 mg/L). However, no significant differences in total fat and carbohydrate oxidation rates were observed between trials. Accordingly, plasma glucose, glycerol, and fatty acid concentrations did not differ between trials. Plasma lactate concentrations were significantly lower in the HCA than in the placebo trial after 30 min of exercise but at the end of the exercise period they did not differ between trials.HCA, even when provided in large quantities, does not increase total fat oxidation in vivo in endurance-trained humans.