Alkoxyl free radical species have been identified by Electron Paramagnetic Resonance (EPR) spectroscopy following exercise and it is postulated that this radical originates from the phospholipid membrane (Davison et al. 2006). However, no conclusive evidence has been provided to confirm this supposition. We therefore tested the hypothesis that exercise-induced oxidative stress is caused by free radical-mediated damage to polyunsaturated fatty acids (PUFA) which can be prevented following ascorbate prophylaxis. Hyperfine coupling constants (HCC) of alpha-phenyl-tert-butylnitrone (PBN)-adducts were measured via room temperature EPR spectroscopy in the venous blood of 12 subjects at rest and following maximal exercise and compared to those observed following room-air incubation (2 hr at 37oC) of L-alpha-phosphatidycholine, linoleic acid, alpha-linolenic acid and arachidonic acid. Subjects also received an acute oral bolus dose of either 1000mg of ascorbic acid or placebo (6 placebo and 6 ascorbic acid), 2 hrs prior to the exercise challenge in a randomised, double-blind, placebo-controlled design, while ascorbic acid was also added to the in vitro model. All adducts exhibited similar HCC [aN 13.6 Gauss (G) and abetaH 1.8G] with the exception of L-alpha-phosphatidycholine [aN1= 13.4G, abetaH1 = 1.6G (37%) and aN2 = 14.9G, abetaH2 = 0.3G (63%)] consistent with the trapping of lipid-derived alkoxyl and oleate radicals respectively. Ascorbic acid pre-treatment ablated free radical formation in both model systems. Furthermore, the decrease in PBN-adduct concentration by ascorbic acid was not an artefact associated with adduct instability and subsequent reduction to an EPR “silent” hydroxylamine. These findings suggest that PBN-trapped free radicals detected following human exercise are likely derived from the oxidation of polyunsaturated fatty acid membranes. Furthermore, clear ex vivo and in vitro evidence suggests that ascorbic acid is an effective antioxidant when required to terminate lipid peroxidation and inhibit the generation of oxygen-centred alkoxyl radicals.