We examined the effects of L-ornithine administration on physical fatigue. In a double-blind, placebo-controlled, 2-way crossover study, 17 healthy volunteers were randomized to L-ornithine (2000 mg/d for 7 days and 6000 mg/d for 1 day as L-ornithine hydrochloride) or placebo for 8 days. The fatigue-inducing physical task consisted of workload trials on a cycle ergometer at fixed workloads for 2 hours on 2 occasions. We found that oral L-ornithine administration promoted lipid metabolism and activated the urea cycle from serum triacylglycerol, ketone bodies, free fatty acids, and blood ammonia level changing. L-ornithine significantly attenuated the subjective feeling of fatigue (measured by visual analog scale at postrecovery) compared with postload (P < .01). Moreover, in female subjects, the subjective feeling of fatigue was significantly lower compared with the placebo group (P < .05). In the physical performance test in female subjects, the decrease in mean speed for 10 seconds maximum pedaling from 0.5- to 3.5-hour trials in the group receiving L-ornithine was smaller than that in the group receiving placebo (P < .05). These results suggest that L-ornithine has an antifatigue effect by increasing the efficiency of energy consumption and promoting the excretion of ammonia. L-ornithine is a free amino acid and is not rich in meats or fish, so it is difficult to obtain amounts of L-ornithine from ordinary meals that would be sufficient to promote the antifatigue effect. We recommend L-ornithine intake as a nutritional supplement in cases of physical fatigue.