The Ross procedure using the inclusion technique with anticommissural plication (ACP) is associated with excellent valve hemodynamics and favorable leaflet kinematics. The objective was to evaluate individual pulmonary cusp's biomechanics and fluttering by including coronary flow in the Ross procedure using an ex vivo three-dimensional-printed heart simulator. Ten porcine and five human pulmonary autografts were harvested from a meat abattoir and heart transplant patients. Five porcine autografts without reinforcement served as controls. The other autografts were prepared using the inclusion technique with and without ACP (ACP and NACP). Hemodynamic and high-speed videography data were measured using the ex vivo heart simulator. Although porcine autografts showed similar leaflet rapid opening and closing mean velocities, human ACP compared to NACP autografts demonstrated lower leaflet rapid opening mean velocity in the right (p = 0.02) and left coronary cusps (p = 0.003). The porcine and human autograft leaflet rapid opening and closing mean velocities were similar in all three cusps. Porcine autografts showed similar leaflet flutter frequencies in the left (p = 0.3) and noncoronary cusps (p = 0.4), but porcine NACP autografts versus controls demonstrated higher leaflet flutter frequency in the right coronary cusp (p = 0.05). The human NACP versus ACP autografts showed higher flutter frequency in the noncoronary cusp (p = 0.02). The leaflet flutter amplitudes were similar in all three cusps in both porcine and human autografts. The ACP compared to NACP autografts in the Ross procedure was associated with more favorable leaflet kinematics. These results may translate to the improved long-term durability of the pulmonary autografts.