The Shear Wave Elastography of muscles can provide real-time information on the stiffness of muscles; however, the difference in stiffness between biceps brachii muscle bellies requires more research. Understanding the variables that affect muscle stiffness will assist in the development of Shear Wave Elastography as a diagnostic tool for muscle stiffness pathologies. This study’s aim is to determine the Shear Wave Velocity of the short and long head of biceps brachii and the change in stiffness with elbow flexion to create a reliable protocol for pathological muscle assessment. The muscle belly of the short and long heads of bilateral biceps brachii of 38 healthy participants were scanned supine with the arm at full extension and at 30° and 60° elbow flexion. A log transform of the SWV was used as the response variable in the regression analysis, and the intraclass correlation coefficient was determined for reliability. The Shear Wave Velocity of the short head was lower than the long head on average. By fitting Bayesian mixed effect regression models to the data, the estimated posterior predictive mean velocities for the short head at full extension, 30°, and 60° were 3.14 m/s, 2.65 m/s, and 2.62 m/s, respectively; and 3.91 m/s, 3.02 m/s, and 3.15 m/s, respectively, for the long head of the biceps brachii. The intraclass correlation coefficients (0.64–0.92) were good to excellent. Shear Wave Elastography can detect the consistent difference in the stiffness of the two muscle bellies of the biceps brachii at multiple elbow angles. The assessment of muscle stiffness with Shear Wave Elastography should consider the morphology of the muscles.