Introduction: Platelet refrigeration decreases the risk of bacterial contamination and may preserve function better than standard-of-care room temperature (RT) storage. Benefits could include lower transfusion-related complications, decreased costs, improved hemostasis in acutely bleeding patients, and extended shelf life. In this study, we compared the effects of 22°C and 4°C storage on the functional and activation status of apheresis platelets. Methods: Apheresis platelets (n = 5 per group) were stored for 5 days at 22°C with agitation (RT) versus at 4°C with agitation (4°C + AG) and without (4°C). Measurements included platelet counts, mean platelet volume, blood gas analytes, aggregation response, thromboelastography, thromboxane B2 and soluble CD40 ligand release, activation markers, and microparticle formation. Results: Sample pH levels were within acceptable limits for storage products (pH 6.2–7.4). Platelet glucose metabolism (P < 0.05), aggregation response (adenosine diphosphate: RT 0; 4°C + AG 5.0 ± 0.8; 4°C 5.6 ± 0.9; P < 0.05), and clot strength (maximum amplitude: RT 58 ± 2; 4°C + AG 63 ± 2; 4°C 67 ± 2; P < 0.05) were better preserved at 4°C compared with RT storage. Refrigerated samples were more activated compared with RT (P < 0.05), although thromboxane B2 (P < 0.05) and soluble CD40 ligand release (P < 0.05) were higher at RT. Agitation did not improve the quality of 4°C-stored samples. Conclusions: Apheresis platelets stored at 4°C maintain more viable metabolic characteristics, are hemostatically more effective, and release fewer proinflammatory mediators than apheresis platelets stored at RT over 5 days. Given the superior bacteriologic safety of refrigerated products, these data suggest that cold-stored platelets may improve outcomes for acutely bleeding patients.