The objective of this study was to validate a recently developed tissue tracking (TT) method for cardiac motion, by comparing it with precise invasive measurements of motion and to prove its capability to reflect moderate hemodynamic changes induced by asynchronous activation. In four open-chest sheep, sono-crystals measured the left ventricle(LV) equator's diameters simultaneously with 2D ultrasound acquisition. The LV was paced either from the posterior or from the lateral wall, just prior to the normal LV activation. Global functional indices were calculated based on the regional motions extracted by the TT method. The correlation coefficient between the shortening of the diameters and the global circumferential strain (GCS) was 0.99 +/- 0.004. The peak GCS differentiated between the pacing modes (paired t test, P < 0.05). The GCS, a measurement closely based on the TT method, followed the precise sono-crystals measurements and reflected moderate hemodynamic changes, thus providing a substantial proof of the TT method's accuracy and clinical value.