Abstract Earlier studies have shown substantial non-uniformity in normal left ventricular (LV) myocardial function, concerning both the degree of shortening and the timing of shortening. We hypothesized that non-uniform LV function may be related to non-uniform prestretch induced by the atrial contraction. Eleven healthy human subjects were studied using MRI myocardial tagging and strain analysis. The amount of circumferential prestretch was assessed in 30 LV segments. Prestretch was defined as the difference in strain between end-diastole (at ECG-R) and diastasis. Furthermore, both the degree of shortening (quantified as peak circumferential shortening, peak systolic shortening rate, and amount of post-systolic shortening) and the timing of shortening (quantified as onset time of shortening and time to peak shortening) were assessed. LV prestretch was found to be non-uniform, with highest values in the lateral wall. The amount of segmental prestretch correlated significantly with peak shortening (r = 0.79), peak shortening rate (r = 0.50), amount of post-systolic shortening (r = 0.67), onset time of shortening (r = -0.57), and time to peak shortening (r = 0.71), P < 0.001 for each of these relations. These relations may be explained by regional differences in wall stress, or by a regional Frank-Starling effect. The correlation between timing of shortening and prestretch demonstrates that mechanical timing is not determined by electrical phenomena alone. In conclusion, regional variation in LV function correlates with the non-uniform prestretch from atrial contraction.