In recent years, novel ultrasound functional imaging (UFI) techniques have been introduced to assess cardiac function by measuring, e.g. cardiac output (CO) and/or myocardial strain. Verification and reproducibility assessment in a realistic setting remain major issues. Simulations and phantoms are often unrealistic, whereas in vivo measurements often lack crucial hemodynamic parameters or ground truth data, or suffer from the large physiological and clinical variation between patients when attempting clinical validation. Controlled validation in certain pathologies is cumbersome and often requires the use of lab animals. In this study, an isolated beating pig heart setup was adapted and used for performance assessment of UFI techniques such as volume assessment and ultrasound strain imaging. The potential of performing verification and reproducibility studies was demonstrated. For proof-of-principle, validation of UFI in pathological hearts was examined. Ex vivo porcine hearts (n = 6, slaughterhouse waste) were resuscitated and attached to a mock circulatory system. Radio frequency ultrasound data of the left ventricle were acquired in five short axis views and one long axis view. Based on these slices, the CO was measured, where verification was performed using flow sensor measurements in the aorta. Strain imaging was performed providing radial, circumferential and longitudinal strain to assess reproducibility and inter-subject variability under steady conditions. Finally, strains in healthy hearts were compared to a heart with an implanted left ventricular assist device, simulating a failing, supported heart. Good agreement between ultrasound and flow sensor based CO measurements was found. Strains were highly reproducible (intraclass correlation coefficients >0.8). Differences were found due to biological variation and condition of the hearts. Strain magnitude and patterns in the assisted heart were available for different pump action, revealing large changes compared to the normal condition. The setup provides a valuable benchmarking platform for UFI techniques. Future studies will include work on different pathologies and other means of measurement verification.