Exact adjustment of the Embden-Meyerhof pathway (EMP) is an important issue in ischemic preconditioning (IP) because an attenuated ischemic lactate accumulation contributes to myocardial protection. However, precise mechanisms of glycolytic flux and its regulation in IP remain to be elucidated. In open chest pigs, IP was achieved by two cycles of 10-min coronary artery occlusion and 30-min reperfusion prior to a 45-min index ischemia and 120-min reperfusion. Myocardial contents in glycolytic intermediates were assessed by high performance liquid chromatographic analysis of serial myocardial biopsies under control conditions and IP. Detailed time courses of metabolite contents allow an in-depth description of EMP regulation during index ischemia using metabolic control analysis. IP reduced myocardial infarct size (control, 90.0 +/- 3.1 versus 5.05 +/- 2.1%; p < 0.001) and attenuated myocardial lactate accumulation (end-ischemic contents, 31.9 +/- 4.47 versus 10.3 +/- 1.26 micromol/wet weight, p < 0.0001), whereby a decrease in anaerobic glycolytic flux by at least 70% could constantly be observed throughout index ischemia. By calculation of flux:metabolite co-responses, the mechanisms of glycolytic regulation were investigated. The continuous deceleration of EMP flux in control myocadium could neither be explained on the basis of substrate availability nor be attributed to regulatory "key enzymes," as multisite regulation was employed for flux adjustment. In myocardium subjected to IP, an even pronounced deceleration of EMP flux during index ischemia was observed. Again, the adjustment of EMP flux was because of multisite modulation without any evidence for flux limitation by substrate availability or a key enzyme. However, IP changed the regulatory properties of most EMP enzymes, and some of these patterns could not be explained on the basis of substrate kinetics. Instead, other regulatory mechanisms, which have previously not yet been described for EMP enzymes, must be considered. These altered biochemical properties of the EMP enzymes have not yet been described.