The aim was to optimize the perfusate composition by including a hemofiltrator to the PhysioHeartplatform for ex situ heart perfusion of porcine slaughterhouse hearts. Fourteen hearts were harvested from Dutch Landrace pigs and slaughtered for human consumption. All hearts were preserved for 4 hours using static cold storage before reperfusion for 4 hours on the PhysioHeart platform. Seven hearts were assigned to the hemofiltration group, where a hemofiltrator was added to the perfusion circuit, while the control group did not receive hemofiltration. In the hemofiltration group, the perfusion fluid was filtrated for 1 hour with a flow of 1 L/hour before reperfusion. After mounting the heart, hemofiltration was maintained at 1 L/hour, and cardiac function and blood samples were analyzed at multiple time points. Preserved cardiac function was defined as a cardiac output >3.0 L/min with a mean aortic pressure >60 mm Hg and a left atrial pressure <15 mm Hg. Hemofiltration resulted in a significantly reduced potassium concentration at all time points (p < 0.001), while sodium levels remained at baseline values (p < 0.004). Furthermore, creatinine and ammonia levels decreased over time. Functional assessment demonstrated a reduced left atrial pressure (p < 0.04) and a reduction of the required dobutamine dose to support myocardial function (p < 0.003) in the hemofiltration group. Preserved cardiac function did not differ between groups. Hemofiltration results in an improved biochemical composition of the whole blood perfusate and preserves cardiac function better during normothermic perfusion based on a reduced left atrial pressure (LAP) and dobutamine requirement to support function.