Introduction: Diastolic dysfunction causes heart failure and decreased exercise tolerance, yet its assessment remains imperfect. We hypothesized that a novel application of two-dimensional speckle tracking echocardiography, which directly measures myocardial strain tissue mechanics, may more accurately quantify diastolic function than currently used measures. Therefore it may more sensitively detect exercise training-induced improvements in diastolic function in subjects enrolled in the NHLBI funded Peripheral Effects of Exercise on Cardiovascular Health Study (STRRIDE II). Methods: Fifteen overweight, sedentary subjects without cardiovascular disease had comprehensive echocardiograms performed before and after an eight-month exercise program. Two-dimensional speckle tracking methods were used to extract left ventricular global longitudinal strain throughout the cardiac cycle, which was analyzed for measures of diastolic function. Changes in strain derived variables were compared to conventional Doppler measures including the ratio of peak early (E) and late (A) diastolic mitral inflow velocities, early inflow to annular velocity ratio (E/e'), and isovolumic relaxation time (IVRT). Results: After exercise training, strain-based variables were unchanged (data not shown), but there were significant improvements in its first derivative, including peak longitudinal early diastolic strain rate (17.4% increase, p=0.03) and early longitudinal peak diastolic strain rate deceleration time (22.3% shorter, p=0.01). In contrast, traditional Doppler variables were unchanged, including E/A ratio (7.2% increase, p=0.29), E/e' (12.2% increase, p=0.13) and IVRT (2.3% shorter, p=0.74). Conclusions: Diastolic function can be assessed using strain analysis of speckle tracking echocardiography. Further, the data obtained are more sensitive in quantifying exercise training-induced improvements in diastolic function than traditional measures. Refinement and application of this novel methodology will enhance our understanding of diastolic function and the positive effects of exercise training.