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Background: Since the greater part of coronary blood flow takes place during the diastolic phase of each cardiac cycle, a time indicator of myocardial perfusion reserve, the ratio of diastolic to systolic duration (D/S ratio), was presented. The objective of this study was to evaluate the accuracy and precision, the biological implication, and the applications of D/S ratio. Methods: Multi-center pragmatic studies evaluating the time indicator of cardiac perfusion reserve were performed. Related experiments, clinical trials, and surveys were conducted at 5 centers. Results: The results showed that the measurement of D/S ratio is both accurate and precise; the mean values of D/S of all of the 3 species studied (human, rabbit, and rat) were greater than 1. These application studies on D/S ratio showed that a close negative correlation existed between D/S ratio and New York Heart Association Functional Classification (NYHA FC) (r = –0.659, p < 0.01); normal persons were mostly distributed at NYHA FC I and at high value of D/S ratio; the patients with cardiovascular disease were mostly at low value of D/S ratio; the difference of D/S between pregnant women with pre-eclampsia and either normal pregnant women or non-pregnant women were significant (p < 0.05); athletes had higher D/S ratio than non-athletes (2.04 ± 0.33 vs 1.82 ± 0.27, p < 0.01). Conclusions: D/S ratio has important biological implication, which is a safe, easy, reliable, and effective indicator, can be used to evaluate fitness levels, served as a pathophysiological marker for screening of cardiovascular disease (CVD), for predicting risk of cardiac events, and for evaluating the severity and prognosis of CVD.