The study shows that critical limb ischaemia (CLI) exacerbates mitochondrial dysfunction and that antioxidant therapies such as N-acetyl cysteine (NAC) still confer protection, even if comorbidities are known to reduce the protective effects of many drugs. Clinical studies using antioxidant strategies have been mostly disappointing and antioxidant therapies are still far from being integrated into treatment algorithms for peripheral arterial disease, but these results suggest that oxidative stress needs to be considered as a target for protective strategies in the setting of CLI. Objectives: The current study was performed in order to determine the influence of hypercholesterolaemia on critical limb ischaemia (CLI) and whether targeting oxidative stress by antioxidant therapies such as N-acetyl cysteine (NAC), considered to be a direct scavenger of reactive oxygen species, could confer muscle protection. Methods: Apolipoprotein E (ApoE)e/e mice (n ¼ 9, 29 weeks old) and their genetic controls ApoEþ/þ mice (n ¼ 9, 29 weeks old) were submitted to sequential right femoral and iliac ligations; the left limb served as control. ApoEþ/þ mice were divided into two groups: Group 1 (n ¼ 4) and Group 2 (n ¼ 5); as well as ApoEe/e mice: Group 3 (n ¼ 3), and Group 4 (n ¼ 6). NAC treatment was administered to Groups 2 and 4 in drinking water. Mice were sacrificed on Day 40 and gastrocnemius muscles were harvested to study mitochondrial respiration by oxygraphy, calcium retention capacity by spectrofluorometry, and production of reactive oxygen species by electron paramagnetic resonance. Results: CLI associated with ApoE deficiency resulted in more severe mitochondrial dysfunction: maximum oxidative capacity and calcium retention capacity were decreased (À42.9% vs. À25.1%, p ¼ .010; and À73.1% vs. À40.3%, p ¼ .003 respectively) and production of reactive oxygen species was enhanced (þ63.6% vs. þ41.4%, p ¼ .03) in ApoEe/e mice compared with ApoEþ/þ mice respectively. Antioxidant treatment restored oxidative capacity, calcium retention capacity and decreased production of reactive oxygen species in both mice strands. Conclusions: In this small murine study, hypercholesterolaemia exacerbated mitochondrial dysfunction, as clinically expected; but antioxidant therapy appeared protective, which is counter to clinical experience. Further work is clearly needed.