Some of the challenges with tissue Doppler measurement include: apparent inconsistency between manufacturers, uncertainty over which part of the trace to make measurements and a lack of calibration of measurements. We develop and test tools to solve these problems in echocardiography laboratories. We designed and constructed an actuator and phantom setup to produce automatic reproducible motion, and used it to compare velocities measured using 3 echocardiographic modalities: M-mode, speckle tracking, and tissue Doppler, against a non-ultrasound, optical gold standard. In the clinical phase, 25 patients underwent M-mode, speckle tracking and tissue Doppler measurements of tissue velocities. In-vitro, the M-mode and speckle tracking velocities were concordant with optical assessment. Of the three possible tissue Doppler measurement conventions (outer, middle and inner line) only the middle line agreed with the optical assessment (discrepancy -0.20 (95% confidence interval -0.44 to 0.03)cm/s, p=0.11, outer +5.19(4.65 to 5.73)cm/s, p<0.0001, inner -6.26(-6.87 to -5.65)cm/s, p<0.0001). All 4 studied manufacturers showed a similar pattern. M-mode was therefore chosen as the in-vivo gold standard. Clinical measurements of tissue velocities by speckle tracking and the middle line of the tissue Doppler were concordant with M-mode, while the outer line significantly overestimated (+1.27(0.96 to 1.59)cm/s, p<0.0001) and the inner line underestimated (-1.81(-2.11 to -1.52)cm/s, p<0.0001). Echocardiographic velocity measurements can be calibrated by simple, inexpensive tools. We found that the middle of the tissue Doppler trace represents velocity correctly. Echocardiographers requiring velocities to match between different equipment, settings or modalities should use the middle line as the “guideline”.