Objectives: Extracellular vesicles (EVs) are heterogeneous membrane vesicles in diameter of 30-5000 nm, that transport proteins, non-coding RNAs (miRNAs), lipids and metabolites. Major populations include exosomes, ectosomes and apoptotic bodies. The purpose of this study was to compare the distribution of EVs obtained under different conditions of differential centrifugation, including ultracentrifugation, with the results developed based on a theoretical model. Methods: Immortalized endothelial cell line that expresses h-TERT (human telomerase) was used to release of EVs: microvascular TIME. EVs were isolated from the culture medium at different centrifugation parameters. The size distribution of the EVs was measured using TRPS technology on a qNano instrument. Surface markers were evaluated using flow cytometry. The isolated EV subpopulations were compared with the theoretical model developed by Livshits. Results: EVs isolated from endothelial cells show strong aggregating properties, which was confirmed by TEM, TRPS imaging and flow cytometry. Conclusions: Obtaining pure EV subpopulations is difficult because of the small differences in the diameter of ectosomes and exosomes, and the strong aggregating properties of EVs.