Purpose: To evaluate the effects of a trail-running race on muscle oxidative function by measuring pulmonary gas exchange variables and muscle fractional O2 extraction. Methods: Eighteen athletes were evaluated before (PRE) and after (POST) a trail-running competition of 32 or 50 km with 2000 or 3500 m of elevation gain, respectively. During the week before the race, runners performed an incremental uphill running test and an incremental exercise by utilizing a 1-leg knee extension ergometer. The knee extension exercise was repeated after the end of the race. During the knee extension test, the authors measured oxygen uptake () and micromolar changes in deoxygenated hemoglobin (Hb)+myoglobin (Mb) concentrations (Δ[deoxy(Hb+Mb)]) on vastus lateralis with a portable near-infrared spectroscopy. Results: was lower at POST versus PRE (−23.9% [9.0%]; P < .001). at POST was lower than at the same workload at PRE (−8.4% [15.6%]; P < .050). Peak power output and time to exhaustion decreased at POST by −23.7% (14.3%) and −18.3% (11.3%), respectively (P < .005). At POST, the increase of Δ[deoxy(Hb+Mb)] as a function of work rate, from unloaded to peak, was less pronounced (from 20.2% [10.1%] to 64.5% [21.1%] of limb ischemia at PRE to 16.9% [12.7%] to 44.0% [18.9%] at POST). Peak Δ[deoxy(Hb+Mb)] values were lower at POST (by −31.2% [20.5%]; P < .001). Conclusions: Trail running leads to impairment in skeletal muscle oxidative metabolism, possibly related to muscle damage from repeated eccentric contractions. In association with other mechanisms, the impairment of skeletal muscle oxidative metabolism is likely responsible for the reduced exercise capacity and tolerance during and following these races.