Time between meals can vary from multiple hours to days within and among species. We investigated the effects of time since feeding on lipid, protein, and carbohydrate oxidation in flying pigeons (Columba livia) by interpreting changes in blood plasma metabolite concentrations and mass during flight. Five pigeons were flown or rested for 4 h after food deprivations of 2, 12, 24, and 48 h. After flight, blood plasma concentrations of uric acid and beta-hydroxybutyrate were elevated over control and preflight values, indicating elevated protein and lipid catabolism during flight. Lipid oxidation, as indicated by changes in beta-hydroxybutyrate concentration, increased more in unfed flying pigeons compared with recently fed flying pigeons and with resting controls. Protein oxidation, as indicated by changes in uric acid concentrations, also positively covaried with feeding time; the covariation was mostly caused by increases in 48-h food-deprived pigeons. Unfed birds lost less mass during a 4-h flight than recently fed birds. We reasoned that recently fed pigeons oxidized more glycogen in flight than pigeons not recently fed; calculated glycogen stores explained 72%-117% of mass loss differences between 2- and 48-h-fed pigeons. Thus, time since feeding was an important determinant of the fuels pigeons used in flight.