Normothermic machine perfusion (NMP) protocols using blood-based solutions are commonly used in the assessment of kidneys prior to transplantation. This procedure is nevertheless limited by blood availability and warrants the search for alternatives. We compared a blood-based solution to a serum-like preservation solution (Aqix) enriched with colloids, with and without red blood cells (RBC). Porcine kidneys retrieved from an abattoir were subjected to 30 min of warm ischemia, followed by 3h of hypothermic oxygenated machine perfusion at 4°C. Subsequently, kidneys (n = 6 per group) were evaluated with NMP for 4h with 5 different solutions: Diluted blood, Aqix with bovine serum albumin (BSA) +/- RBC's; or Aqix with Dextran 40 +/- RBC's. Throughout NMP, markers of renal function and tubular metabolism were favorable in RBC's groups. Addition of RBC's resulted in a 4- to 6-fold higher oxygen consumption rates. Controls had significantly higher ATP levels post-NMP, exhibited decreased production of oxidative stress markers, and had the highest creatinine clearance. In conclusion, this study shows that addition of RBC's during NMP reduced renal injury, improved function, and was associated with increased renal metabolism. Although the RBC-BSA-supplemented Aqix solution was also able to support metabolism and renal function, a blood-based perfusion solution remains superior.