Technical Abstract: Metabolites of the cytochrome P450 pathway may contribute to vasodilation of the vasculature of skeletal muscle during exercise. We determined effects of exercise intensity and duration on plasma concentrations of specific metabolites in the epoxyeicosatrienoic acid family. This allowed us to determine the threshold workload, optimal workload, and time period required to produce increases in plasma concentrations of these vasoactive substances. Fourteen healthy volunteers performed maximal exercise testing on a bicycle ergometer during visit 1. On separate days subjects performed exercise testing for 20 min at 30%, 60%, and 80% of their maximal workload. The last day consisted of 40min of exercise at 60% workload. Venous blood was obtained before, during and after exercise for analysis. Compared to baseline, significant increases were observed during the 80% workload at 20 min: 14,15 DHET (0.77±0.21 vs. 0.93±0.27 pmol/ml) and at 2 min post exercise: 11,12 DHET (0.64±0.22 vs. 0.71±0.24 pmol/ml). Mean 40 min values at 60% workload were: 14,15 DHET (0.79±0.22 vs. 0.91±0.31 pmol/ml)and at 2 min post: 14,15 EET (0.12± 0.06 vs 0.21 ±0.16 pmol/ml). Results suggest that these metabolites are released during short term high intensity or long term moderate intensity exercise.