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Although there have been many investigations of the beneficial effects of both exercise and amino acids (AAs), little is known about their combined effects on the single-dose ingestion of AAs for lipid metabolism during exercise. We hypothesize that taking a specific combination of AAs implicated in glucagon secretion during exercise may increase fat metabolism. We recently developed a new mixture, d–AA mixture (D-mix), that contains arginine, alanine, and phenylalanine to investigate fat oxidation. In a double-blind, placebo-controlled crossover study, 10 healthy male volunteers were randomized to ingest either D-mix (3 g/dose) or placebo. Subjects in each condition subsequently performed a physical task that included workload trials on a cycle ergometer at 50% of maximal oxygen consumption for 1 hr. After oral intake of D-mix, maximum serum concentrations of glycerol (9.32 ± 6.29 mg/L and 5.22 ± 2.22 mg/L, respectively; p = .028), free fatty acid level (0.77 ± 0.26 mEq/L and 0.63 ± 0.28 mEq/L, respectively; p = .022), and acetoacetic acid levels (37.9 ± 17.7 μmol/L and 30.3 ± 13.9 μmol/L, respectively; p = .040) were significantly higher than in the placebo groups. The area under the curve for glucagon during recovery was numerically higher than placebo (6.61 ± 1.33 μg/L • min and 6.06 ± 1.23 μg/L • min, respectively; p = .099). These results suggest that preexercise ingestion of D-mix may stimulate fat metabolism. Combined with exercise, the administration of AA mixtures could prove to be a useful nutritional strategy to maximize fat metabolism.