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Platelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) and room temperature-stored platelets (RSP) in vitro, in vivo, and post-transfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared to RSP. We found significantly reduced GPVI levels after cold exposure of 5-7 days. After transfusion in humans, CSP were mostly equivalent to RSP yet aggregated significantly less to the GPVI agonist collagen. In a mouse model of platelet transfusion, we found a significantly lower response to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of CSP and highlight the need for thorough investigations of this product. (NCT03787927)