Extracellular vesicles (EVs) may serve as biomarkers for multiple purposes, samples of plasma and serum offer in the form of liquid biopsy a relatively non-invasive means to obtain EV-mediated molecular information. High throughput sampling and analytics for the search of EV-borne biomarkers is facilitated by the availability of biobanks, but the samples in biobanks have not been collected with EVs in mind. In addition to anticoagulation, pre-analytical steps of EV-research as e.g. agitation and prolonged incubation cause ex vivo platelet activation and vesiculation. This study analyses the effects of common anticoagulation (citrate, EDTA and ACD) and clotting on EV-samples from plasma/serum pools that had been equally treated during the blood collection and utilizing the currently approved preanalytical steps. Blood samples were obtained from healthy, fasting volunteers. Volunteers had not used any medication for the previous 7 days. Pools of samples were analyzed with quantitative label-free proteomics using ultra-definition MSE. Statistical analyzes were performed to identify proteins distinguishing different sample types. With two or more unique proteins per identification, 163 proteins were quantified. A clear separation between sample types was seen in principal component analysis. 42 proteins showed statistically significant difference between sample types. The set of proteins was studied further with network, pathway and protein-protein interaction analyses, and found to participate in e.g. cell growth and maintenance.