High cardiac energy demand relies on lipid & glucose oxidation, depending on oxygen availability [1]. The relative importance of functional capillary supply or ability to switch between substrates to preserve cardiac performance is currently unclear, but may be critically important in conditions such as diabetes that are characterized by a reduced metabolic flexibility through the loss of insulin sensitivity [2]. The role of arteriole perfusion in selection of substrates was established before and after infusion of polystyrene microspheres (mean diameter 15.0±0.2µm) to mimic capillary rarefaction due to microvascular disease. The effect of acute loss of functional capillary supply on palmitate and glucose metabolism together with function was determined in normal working hearts from male rats. Random blockade of coronary arterioles by microspheres decreased rate-pressure product (RPP; R2=0.82) in a dose-dependent manner, and halved peak developed pressure (P<0.01), in perfused-working hearts [3] from control and STZ-diabetic rats. Palmitate oxidation halved (P<0.001) with a similar increase in glucose oxidation (P<0.01) for control hearts, but in diabetic hearts palmitate oxidation was 2.5-fold higher (P<0.001) with unchanged glucose oxidation (ns). Arteriole occlusion decreased the density of patent capillaries by 40% (P<0.001) and increased capillary supply area by 60% (P<0.01). There was a more heterogeneous distribution of functional capillaries in the myocardium following microsphere infusion (CV=19.15% untreated vs. 25.80% occluded). Calculated myocardial PO2 was sensitive to occlusion of arterioles leading to a decrease in oxygen consumption, and an increase in the fractional area of hypoxic regions. Graded loss of arteriole supply thus led to a reduction in both coronary flow and cardiac work, and metabolism was also decreased as a consequence. Interestingly, hearts demonstrate the capacity to switch between substrates, from palmitate to glucose, to maximise oxygen efficiency and sustain cardiac work. Our data suggests that the decline in performance noted for the heart in chronic diabetes may not be metabolic in origin but could result from the secondary consequence of diabetes, namely capillary rarefaction and microvascular disease leading to a regional inadequacy in oxygenation of the myocardium. This supports the importance of preventing arteriole loss as a consequence of microvascular disease.