Myocardial ischemia-reperfusion injury is common during cardiac procedures. Glutamine may protect the myocardium by preserving metabolic substrates. Glutamine (0.52 g·kg⁻¹) or Ringer's lactate solution (control group) was administered intraperitoneally to 63 Sprague-Dawley rats at 4 or 18 hours prior to experimental ischemia and reperfusion. The hearts were excised and perfused on an isolated working heart model, exposed to global ischemia for 15 min and reperfusion for 1 hour. Left atrial pressure, mean aortic pressure, cardiac flow, coronary flow, and aortic output were measured 15 min before ischemia and every 15 min during reperfusion. There was significantly better cardiac output in the glutamine pretreated groups. Pretreatment at 4 hours before the experiment was superior to pretreatment at 18 hours, with better maintenance of cardiac output and coronary flow. The enhanced protective effect of pretreatment at 4 hours highlights the importance of timing, and suggests a potential clinical benefit.