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Aim: To test the agreement between strain measurements with 3D echocardiaography and sonomicrometer in an in vitro setup using a left ventricular phantom.Methods: A polyvinyl alcohol phantom of the left ventricle was connected to a stepping motor controlled volume pump and submerged in a water container. Using a Vivid E9 scanner and a 4V probe (GE Vingmed Ultrasound, Horten, Norway), 3D ultrasound recordings were acquired at 15 different stroke volumes (10 to 150 ml), all at a heart rate of 60 beats/min. As a reference method, 8 sonomicrometer crystals were implanted into the phantom, monitoring changes in longitudinal, circumferential and radial lengths. For each situation, single beat as well as several multi beat recordings of 6 cycles were acquired at several frame rates (FR). 3D strain curves were generated using EchoPac BT11 (GE Vingmed Ultrasound). Peak strain values were compared with sonomicrometry to evaluate the accuracy of 3D speckle tracking strain values for different FR and volumes, using correlation coefficients and Bland-Altman analysis.Results: The table shows the comparison of longitudinal and circumferential strain at different frame rates. Sonomicrometry values, spanning from -1.86 % to -22.15 % in the circumferential direction, correlated well to stroke volume (r = −0.99). Multi beat acquisition improved accuracy. Best correlation and smallest limits of agreement (±1.96SD) were obtained with a FR of 36.6 frames/sec (FPS).Conclusions: Multi beat gave excellent agreement with sonomicrometer, best agreement were obtained at 36.6 FPS, indicating a possible optimal FR for 3D strain measurement lower than for corresponding 2D strain.