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The interest around circulating extracellular vesicles and their cargo in diagnostics has greatly increased; however, several pre-analytical variables affect their determination. In this study, we investigated the effects of sample matrix, processing, and plasma storage delay and temperature on extracellular vesicles and their miRNA content. Blood was collected from 10 male volunteers in dipotassium ethylendiaminotetraacetate-coated tubes (K EDTA), either with plasma-preparation tube (PPT) or without (K2E) gel separator. A stepwise centri