Background: Subnormothermic oxygenated machine perfusion has been shown to improve kidney preservation in the preclinical setting. However, whether oxygenated perfusion without an oxygen carrier provides superior protection over standard hypothermic machine perfusion at 4 °C in human kidneys has not been established. Methods: In this study, we assessed whether oxygenated University of Wisconsin (UW) solution perfusion at 22 °C provides improved protection compared with conventional hypothermic machine perfusion (HMP) at 4 °C using a paired human kidney model. Five pairs of discarded human kidneys were recruited for the study. For each donor pair, one kidney was randomized to 12 h oxygenated UW perfusion at 22 °C and the contralateral kidney was perfused at 4 °C with a clinically relevant kidney perfusion pump. After preservation, both kidneys were reperfused with whole blood at 37 °C for 4 h. Hemodynamic and functional parameters were assessed during preservation and reperfusion, including vascular resistance, renal blood flow, creatinine clearance, blood urea nitrogen, electrolyte levels, tissue oxygenation, and urine output. Histological injury was evaluated and a panel of kidney injury biomarkers were also measured. Results: Vascular resistance during the preservation period was significantly lower in the kidneys preserved at 22 °C compared to the HMP group, indicating improved flow dynamics. However, the improved vascular resistance observed during 22 °C preservation did not translate into superior post-reperfusion kidney function or tissue protection compared with 4 °C HMP. Renal blood flow, creatinine clearance, tissue oxygenation, cytochrome c release and malondialdehyde (MDA) levels were comparable between groups, while kidneys preserved at 22 °C had significantly higher acute tubular necrosis scores, higher blood urea nitrogen (BUN) levels, increased calbindin, glutathione S-transferase A1, monocyte chemoattractant protein-1 levels, and lower epidermal growth factor levels. Conclusion: These data demonstrate that oxygenated UW perfusion at 22 °C fails to provide superior protection to conventional HMP at 4 °C in discarded human kidneys transplantation model.