BACKGROUND: Conventional platelet (PLT) storage at room temperature under continuous agitation results in a limited shelf life (5 days) and an increased risk of bacterial contamination. However, both of these aspects can be ameliorated by cold storage. Preliminary work has suggested that PLTs can be cold stored for up to 3 weeks, while preserving their metabolic activity longer than in PLTs stored at room temperature. As such, in the present study, we hypothesized that the metabolic phenotypes of PLTs stored at 4°C for 3 weeks could be comparable to that of room temperature–stored PLTs at 22°C for 5 days. Study Design and Methods: Metabolomics analyses were performed on nine apheresis PLT concentrates stored either at room temperature (22°C) for 5 days or refrigerated conditions (4°C) for up to 3 weeks. RESULTS: Refrigeration did not impact the rate of decline in glutamine or the intracellular levels of Krebs cycle metabolites upstream to fumarate and malate. It did, however, decrease oxidant stress (to glutathione and purines) and slowed down the activation of the pentose phosphate pathway, glycolysis, and fatty acid metabolism (acyl-carnitines). CONCLUSION: The overall metabolic phenotypes of 4°C PLTs at Storage Day 10 are comparable to PLTs stored at 22°C at the end of their 5-day shelf life, while additional changes in glycolysis, purine, and fatty acid metabolism are noted by Day 21.