Aim To investigate whether transient myocardial ischemia might cause protein aggregation, and to observe whether heat shock pretreatment and ischemic preconditioning could alleviate protein aggregation. Methods Myocardial ischemial-reperfusion injury model was prepared by ligation of left descending anterior coronary artery for 15 minutes then released for various durations in rats. Protein aggregates induced by ischemia-reperfusion injury in cardiomyocytes were observed by using ethanolic phosphotungstic acid (EPTA) electron microscopy (EM). The influence of heat shock protein 70 (HSP70) and αB-crystallin on above protein damage was further investigated by immunoelectron microscopy. Results ①Myocardial ischemia-reperfusion resulted in protein aggregation, which appeared at 30 min of reperfusion, peaked at 4 h of reperfusion, decreased from 24 h of reperfusion and restored to normal at 72 h of reperfusion after 15 min of ischemia. ②Heat shock pretreatment (rectal temperature 42℃ for 15 min then recovery for 24 h) and ischemic preconditioning (ischemia for 3 min then reperfusion for 10 min ) significantly decreased myocardial protein aggregates induced by 15 min of ischemia and 4 h of reperfusion and facilitated the restoration of myocardial protein damage. ③HSPs played key roles in the myocardial protection of heat shock pretreatment and ischemic preconditioning. In the present study, we found that HSP70 and αB-cystallin co-localized with protein aggregates in heat shock pretreatment and ischemic preconditioning groups. Conclusion This study, for the first time, demonstrated the formation, intracellular distribution and dynamic patterns of myocardial protein aggregates induced by ischemia-reperfusion injury, and found that heat shock pretreatment and ischemic preconditioning alleviated the formation of myocardial protein aggregates.