Lipoxins (LX) are lipoxygenase-derived eicosanoids with potent anti-inflammatory activities and vascular bed-dependent vasodilatory actions. LX can be formed in vitro and in vivo in a number of conditions, and we have reported that immunoreactive LXA₄ (iLXA₄) is physiologically excreted with human urine. Using a recently developed LX extraction method coupled to an ELISA, we examined whether iLXA₄ excretion was modified by strenuous exercise, which is known to trigger potential LX-forming events. Maximal exertion significantly increased iLXA₄ urinary excretion in nine healthy volunteers (0.061 ± 0.023 vs. 0.113 ± 0.057 ng/mg creatinine; P = 0.028). iLXA₄ levels returned to baseline after 6 h and increased, although at a smaller extent, after 24 h. A significant correlation (r = 0.988) was denoted between iLXA₄ ELISA measurements and reversed-phase high-performance liquid chromatography quantitation of a previously described urinary tetraene, confirming its LXA₄-related nature. These findings show for the first time that an increase in excretion of LXA₄-related compounds can be observed in response to strenuous exercise. This may be the reflection of an enhanced LX biosynthesis, which may represent a safeguard mechanism that keeps the inflammatory reaction triggered by physical stress under control.