p-Synephrine, the principal alkaloid of bitter orange (Citrus aurantium), is widely used in dietary supplements for weight loss due to its purported effect of increasing fat oxidation. However, there is a paucity of scientific information about its effectiveness in enhancing fat oxidation during exercise. The aim of this investigation was to determine the effect of an acute dose of p-synephrine on substrate oxidation during prolonged and constant intensity exercise. In a double-blind and randomized experiment, 14 healthy subjects performed two acute experimental trials after ingesting either p-synephrine (3mgkg-1) or a placebo (cellulose). Energy expenditure and fat oxidation rates were continuously measured by indirect calorimetry during 1h of continuous cycling at Fatmax, the intensity that induces maximal fat oxidation rate. In comparison to the placebo, energy expenditure during 1h of cycling remained unchanged with p-synephrine (698 ± 129 vs. 686 ± 123kcal, P = 0.08). However, p-synephrine increased whole-body fat oxidation (33.6 ± 10.4 vs. 37.3 ± 9.8g, P < 0.01) while also reducing carbohydrate oxidation (99.5 ± 30.4 vs. 85.0 ± 28.4g, P < 0.01). However, the magnitude of the shift on substrate oxidation induced by p-synephrine was small. Acute ingestion of p-synephrine augments fat oxidation during prolonged and constant-intensity exercise.