Blood-derived microRNA signatures have emerged as powerful biomarkers for predicting and diagnosing cardiovascular disease, cancer, and metabolic disorders. Platelets and platelet-derived microvesicles are a major source of microRNAs. We have previously shown that the inappropriate anticoagulation and storage of blood samples causes substantial platelet activation that is associated with the release of platelet-stored molecules into the plasma. However, it is currently unclear if circulating microRNA levels are affected by artificial platelet activation due to suboptimal plasma preparation. To address this issue, we used a standardized RT-qPCR test for 12 microRNAs (thrombomiR®, TAmiRNA GmbH, Vienna, Austria) that have been associated with cardiovascular and thrombotic diseases and were detected in platelets and/other hematopoietic cells. Blood was prevented from coagulating with citrate–theophylline–adenosine–dipyridamole (CTAD), sodium citrate, or ethylenediaminetetraacetic acid (EDTA) and stored for different time periods either at room temperature or at 4 °C prior to plasma preparation and the subsequent quantification of microRNAs. We found that five microRNAs (miR-191-5p, miR-320a, miR-21-5p, miR-23a-3p, and miR-451a) were significantly increased in the EDTA plasma. Moreover, we observed a time-dependent increase in plasma microRNAs that was most pronounced in the EDTA blood stored at room temperature for 24 h. Furthermore, significant correlations between microRNA levels and plasma concentrations of platelet-stored molecules pointed towards in vitro platelet activation. Therefore, we strongly recommend to (i) use CTAD as an anticoagulant, (ii) process blood samples as quickly as possible, and (iii) store blood samples at 4 °C whenever immediate plasma preparation is not feasible to generate reliable data on blood-derived microRNA signatures.