We examined the effect of dietary supplementation with L-arginine on breath condensate VEGF, exhaled nitric oxide (NO), plasma erythropoietin, symptoms of acute mountain sickness, and respiratory related sensations at 4,342 m through the course of 24 h in seven healthy male subjects. Serum L-arginine levels increased in treated subjects at time 0, 8, and 24 h compared with placebo, indicating the effectiveness of our treatment. L-arginine had no significant effect on overall Lake Louise scores compared with placebo. However, there was a significant increase in headache within the L-arginine treatment group at 12 h compared with time 0, a change not seen in the placebo condition between these two time points. There was a trend (p = 0.087) toward greater exhaled NO and significant increases in breath condensate VEGF with L-arginine treatment, but no L-arginine effect on serum EPO. These results suggest that L-arginine supplementation increases HIF-1 stabilization in the lung, possibly through a NO-dependent pathway. In total, our observations indicate that L-arginine supplementation is not beneficial in the prophylactic treatment of AMS.