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Standardizing ultrasonic bulk wave velocity measurements on cortical bone is needed.Effects of multiple factors on the resulting stiffness are investigated.A methodology is recommended to comply a protocol for measuring bone stiffness.Elasticity assessment based on bulk wave velocity (BWV) measurements is the most popular technique to characterize the anisotropic stiffness tensor in cortical bone. Typically, a cuboid bone specimen is cut with its sides along the different anatomical directions. Then, the velocity of shear and longitudinal waves propagating along different directions are assessed, from which stiffness coefficients are calculated. Despite the importance of obtaining accurate elasticity values for bone research, there is no generally accepted protocol to measure BWV and the precision of the technique has been seldom investigated. The purpose of this work is to critically assess the method to measure BWV on cuboid specimens in terms of ultrasound frequency, specimen size and signal processing technique. In this study, we measured polycarbonate specimens of different dimensions and 55 human bone specimens with different transducers using frequencies ranging from 2.25 to 10 MHz and 1–5 MHz for longitudinal and shear waves, respectively. We compared four signal processing methods to detect the wave arrival time. The main results are that, (1) the measurement of shear waves is more complex than that of longitudinal wave, being less precise and more sensitive to sample size; (2) the estimated stiffness depends on the signal processing technique used (up to 10% variation for shear coefficients of bone); and (3) bone stiffness assessed from BWV using the first arrival of the signal to determine the time-of-flight is not different from stiffness assessed using resonant ultrasound spectroscopy (RUS). These results evidence that the measurement method can have an effect on the stiffness values estimates and hence, a well-defined protocol is needed to accurately measure bone stiffness coefficients based on BWV.