Updated project metadata. Ex vivo assays of platelet function critically inform mechanistic and clinical studies of hemostasis and thrombosis, where effects of divergent blood processing methods on platelet composition are apparent but remain unspecified. Parallel blood samples were collected from healthy human donors into sodium citrate, acid citrate dextrose, EDTA and heparin, and processed over an extended time course for physiological and biochemical experiments, including platelet proteome quantification with multiplexed tandem mass tag (TMT) labeling and high-resolution Tribrid mass spectrometry (MS). We evaluated how different blood anticoagulation options and processing times affect platelet protein content ex vivo. Following platelet isolation, TMT-MS quantified 3,358 proteins amongst all prepared platelet samples. Altogether, >400 proteins were differentially abundant in platelets isolated from blood processed at 24 h vs. 1 h post-phlebotomy, including sets of proteins pertinent to membrane trafficking and exocytosis.