The effect of mild (15min) and moderate (mod; 40min) global ischemia (GI) upon substrate oxidation, oxygen consumption (MVO2) and cardiac output (CO) during reperfusion was investigated. Hearts were perfused with buffer containing 3% BSA, red blood cells, insulin (0.05 mU/mL). β-hydroxybutyrate (β-HB; 0.2 mM), uniformly labeled 13C-palmitate (ffa; 0.8 mM), l-13C-glucose (GLU; 4 mM) and 3-13C-lactate (LAC; 0.8 mM). Isotopomer analysis, MVO2 and perfusate differences were used to determine the simultaneous contribution of the four substrates to TCA cycle turnover. GI hearts were further subdivided into LAC oxidizing and LAC producing. There was a redistribution of substrate oxidation following GI (see Table). No change was detected between control and mild GI in MVO2 (2.2±0.2, vs 1.6±0.1 μmoles O2/min/g) or CO (86±8 ml/min vs 78±7 ml/min). A decrease in MVO2 (0.40±0.3 μmoles/min/g) and CO (19±10 ml/min) was seen following mod GI. These results suggest in hearts perfused with a mixture of substrates, substrate oxidation is dependent upon GI duration. Oxidation rates in control and ischemic hearts (nmoles /min/g wet; mean±SEM). Group N ffa β-HB GLU LAC Control 8 320±10 150±30 20±10 210±30 mild GI 3a 19013 110±40 80±4 0 mild GI 3b 250±6 130±50 50±30 290±30 modGI 3a 5±1 20±2 30±5 0 mod GI 1b 3 20 10 40 a Net lactate production. b Net lactate utilization.