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Despite the availability of multiple solutions for assessing myocardial strain by ultrasound, little is currently known about the relative performance of the different methods. In this study, we sought to contrast two strain estimation techniques directly (speckle tracking and elastic registration) in an in vivo setting by comparing both to a gold standard reference measurement. In five open-chest sheep instrumented with ultrasonic microcrystals, 2-D images were acquired with a GE Vivid7 ultrasound system. Radial (εRR), longitudinal (εLL), and circumferential strain (εCC) were estimated during four inotropic stages: at rest, during esmolol and dobutamine infusion, and during acute ischemia. The correlation of the end-systolic strain values of a well-validated speckle tracking approach and an elastic registration method against sonomicrometry were comparable for εLL (r=0.70 versus r=0.61 , respectively; p=0.32) and εCC (r=0.73 versus r=0.80 respectively; p=0.31). However, the elastic registration method performed considerably better for εRR (r=0.64 versus r=0.85 respectively; p=0.09). Moreover, the bias and limits of agreement with respect to the reference strain estimates were statistically significantly smaller in this direction (p <; 0.001). This could be related to regularization which is imposed during the motion estimation process as opposed to an a posteriori regularization step in the speckle tracking method. Whether one method outperforms the other in detecting dysfunctional regions remains the topic of future research.