PURPOSE: Splanchnic hypoperfusion is a physiological phenomenon during strenuous exercise. It has been associated with gastrointestinal symptoms, intestinal injury, and may hamper athletic performance. We hypothesized that L-citrulline supplementation improves splanchnic perfusion and decreases intestinal injury by enhancing arginine availability. The aim of this study was to determine the effect of L-citrulline intake on splanchnic perfusion, intestinal injury and barrier function during exercise. METHODS: In this randomized, double-blinded crossover study, ten men cycled for 60 min at 70% of their maximum workload after L-citrulline (10 g) or placebo (L-alanine) intake. Splanchnic perfusion was assessed using gastric air tonometry. Sublingual microcirculation was evaluated by sidestream dark-field imaging. Plasma amino acid levels and intestinal fatty-acid binding protein (I-FABP) concentrations, reflecting enterocyte damage, were assessed every 10 min. Urinary excretion of sugar probes was measured to evaluate intestinal permeability changes. RESULTS: Oral L-citrulline supplementation enhanced plasma citrulline (1840.3+/-142.3 muM) and arginine levels (238.5+/-9.1 muM) compared to placebo (45.7+/-4.8 muM and 101.5+/-6.1 muM, respectively; P<0.0001), resulting in increased arginine availability. Splanchnic hypoperfusion was prevented during exercise following L-citrulline ingestion (reflected by unaltered gapg-apCO2 levels), whereas gapg-apCO2 increased with placebo treatment (P<0.01). Accordingly, L-citrulline intake resulted in an increased number of perfused small sublingual vessels compared with placebo (7.8+/-6.0 vs. -2.0+/-2.4; P=0.06). Furthermore, plasma I-FABP levels were attenuated during exercise following L-citrulline supplementation compared to placebo (AUC0-60min -185+/-506 vs. 1318+/-553 %; P<0.01). No significant differences were observed for intestinal permeability. CONCLUSION: Pre-exercise L-citrulline intake preserves splanchnic perfusion and attenuates intestinal injury during exercise in athletes compared to placebo, probably by enhancing arginine availability. These results suggest that oral L-citrulline supplementation is a promising intervention to combat splanchnic hypoperfusion-induced intestinal compromise.