The purpose of this experiment was to test the effects of prolonged strenuous running on the myocardium. Male SD rats weighting 270-320g were divided into a control (without exercise), immediately, 3-4, 24 and 100 hours post running group. The exercise volume for all running group was 4000-4500 meters, at a speed of 20-25 meter/min, for 3 to 4 hours. Only the rats had completed the exercise criteria were used for this study (About 75 out 150 rats reached this criteria). The myocardial lysosomal enzymes ACP (acid phosphatase) and beta-glucuronidase (beta-Glu), and myocardial mitochondrial calcium were tested in different groups. The blood CK, CK-mb, CK-mm isoforms, LDH, LDH isoenzymes, endothelin, atrial natriuretic factors, acid phosphatase, and beta-glu were also tested. The average body weight loss of rats after running was 17g (6% body weight), and blood glucose decreased 15.8%. Results indicated that the myocardial ACP, beta-Glu, and mitochondrial calcium were significant increased immediately post exercise, and reached peak values 3 to 24 hours post exercise (p<0.01). The blood ACP, beta-Glu, CK, CK isoenzymes and isoforms of different isoenzymes. LDH, LDH isoenzymes and endothelin also demonstrated a delayed elevation during post exercise period(p<0.05). These results indicate that strenuous prolonged exercise may induce myocardial damage and delayed onset myocardial damage.