We investigated the antifatigue effect of royal jelly (RJ), which had been stored at -20 degrees C from immediately after collection, in male Std ddY mice. The mice were accustomed to swimming in an adjustable-current swimming pool, then subjected to forced swimming five times during 2 wk, and the total swimming period until exhaustion was measured. They were separated into three groups with equal swimming capacity, which were administered RJ, RJ stored at 40 degrees C for 7 d (40-7d RJ), or the control solution including casein, cornstarch, and soybean oil before swimming. All mice were forced to swim for 15 min once; then the maximum swimming time to fatigue was measured after a rest period. The swimming endurance of the RJ group significantly increased compared with those of the other groups. The mice in the RJ group showed significantly decreased accumulation of serum lactate and serum ammonia and decreased depletion of muscle glycogen after swimming compared with the other groups, whereas there was no significant difference between the 40-7d RJ group and the control group in these parameters after swimming. A quantitative analysis of constituents in RJ showed that 5 7-kDa protein, which we previously identified as a possible freshness marker of RJ, was specifically degraded in RJ stored at 40 degrees C for 7 d, whereas the contents of various vitamins, 10-hydroxy-2-decenoic acid, and other fatty acids in RJ were unchanged. These findings suggest that RJ can ameliorate the physical fatigue after exercise, and this antifatigue effect of RJ in mice seems to be associated with the freshness of RJ, possibly with the content of 5 7-kDa protein.