Cartilage breakdown, inflammation and pain are hallmark symptoms of osteoarthritis, and autologous blood products such as citrate-anticoagulated platelet-rich plasma (CPRP) or hyperacute serum (hypACT) have been developed as a regenerative approach to rebuild cartilage, inhibit inflammation and reduce pain. However, mechanisms of action of these blood derivatives are still not fully understood, in part due to the large number of components present in these medical products. In addition, the discovery of extracellular vesicles (EVs) and their involvement in intercellular communication mediated by cargo molecules like microRNAs (miRNAs) opened up a whole new level of complexity in understanding blood products. In this study we focused on the development of an isolation protocol for EVs from CPRP and hypACT that can also deplete lipoproteins, which are often co-isolated in EV research due to shared physical properties. Several isolation methods were compared in terms of particle yield from CPRP and hypACT. To gain insights into the functional repertoire conveyed via EV-associated miRNAs, we performed functional enrichment analysis and identified NFκB signaling strongly targeted by CPRP EV miRNAs, whereas hypACT EV miRNAs affect IL6- and TGFβ/SMAD signaling.