Trehalose increases blood glucose levels slowly and induces a slight insulin response. The present study aimed to study the effect of trehalose on prolonged exercise performance. The participants were 12 healthy men (age: 21.3 ± 0.9 y). After an overnight fast (12 h), they first exercised with a constant load (intensity: 40% O2peak) for 60 min using a bicycle ergometer. They continued to exercise with a constant load (40% O2peak) for 30 min between four sets of the 30-s Wingate test. After the 1st set, each participant ingested 500 mL water (control), 8% glucose, or 8% trehalose in three trials. These three trials were at least one week apart and were conducted in a double-blind and randomized crossover manner. Blood was collected for seven biochemical parameters at 12 time points during the experiment. The area under the curve of adrenaline after ingestion of trehalose was significantly lower than that for water and tended to be lower than that for glucose in the later stage of the exercise. Lower secretion of adrenaline after a single dose of 8% trehalose during prolonged exercise reflected the preservation of carbohydrates in the body in the later stage of the exercise. In conclusion, a single ingestion of trehalose helped to maintain prolonged exercise performance.