Patients with chronic heart failure (CHF) and chronic obstructive pulmonary disease (COPD) suffer from exercise intolerance and may also be chronically hypoxic. It is not clear whether this exercise intolerance is due to skeletal muscle dysfunction or changes in oxygen delivery (DO2). Acclimation to chronic hypoxia involves, increased haematocrit and angiogenesis, which may normalise DO2, and changes in skeletal muscle. We hypothesised that changes in skeletal muscle that occur with 3 weeks acclimation to chronic hypoxia (CH; 12% O2) would lead to greater skeletal muscle fatigue. Therefore, the objective of this study was to investigate the effect of changing DO2 by isovolaemic haemodilution on muscle fatigue, in normoxic (N) and CH male Wistar rats. CH rats were housed at 12% O2 in a hypoxic chamber for three weeks before experiments and breathed 12% O2 throughout the following protocol. In all animals, anaesthesia was induced using isoflurane, 4% in O2, and maintained with a continuous infusion of Alfaxalone (13.5-18.0 mg.kg-1.hr-1 I.V.). Arterial blood pressure, femoral blood flow and tension in the extensor digitorum longus (EDL) muscle were recorded before and during peroneal nerve stimulation (15Hz) to evoke EDL contraction for five 3 minute periods; baselines were allowed to recover between stimulations. In the CH group (n=4) and one group of N animals (NHD; n=5) isovolaemic haemodilution was performed between each period of stimulation to reduced DO2. A further group of N animals acted as a time control (TC; n=5). In the TC group the responses to the five 3 minute muscle contractions were consistent (tension time index ∼1200g.s-1 and fatigue ~40%). CH rats fatigued ∼10% more than NHD rats at matched DO2 achieved by isovolaemic haemodilution. It is therefore possible that skeletal muscle adaptations, evoked by chronic hypoxia, play a greater role in the mechanisms limiting exercise tolerance than reductions in DO2.