Ex situ heart perfusion (ESHP) has been developed to decrease cold ischemia time and allow metabolic assessment of donor hearts prior to transplantation. Current clinical ESHP systems preserve the heart in an unloaded condition and only evaluate the cardiac metabolic profile. In this pilot study we performed echocardiographic functional assessment using two alternative systems for left ventricular (LV) loading: pump supported afterload working mode (SAM) and passive afterload working modes (PAM). Six hearts were procured from male Yorkshire pigs. During cold ischemia, hearts were mounted on our custom made ESHP circuit and a 3D-printed enclosure for the performance of echocardiography with a standard TEE probe. Following perfusion with Langherdorf mode of the unloaded heart, the system was switched into different working modes to allow LV loading and functional assessment: pump supported (SAM) and passive (PAM). Echocardiographic assessment of left ventricular function in the donor hearts was performed in vivo and at 1 h of ESHP with SAM, after 4.5 h with PAM and after 5.5 h with SAM. We obtained good quality epicardial echocardiographic images at all time points allowing a comprehensive LV systolic assessment. All indices showed a decrease in LV systolic function throughout the trial with the biggest drop after heart harvesting. We demonstrated the feasibility of echocardiographic functional assessment during ESHP and two different working modes. The expected LV systolic dysfunction consisted of a reduction in EF, FAC, FS, and strain throughout the experiment with the most significant decrease after harvesting.