Extracellular vesicles (EVs) play central physiological and pathophysiological roles in intercellular communication. Biomarker studies addressing disorders such as cardiovascular diseases often focus on circulating microRNAs (miRNAs) and may, depending on the type of disease and clinic routine, utilise patient specimens sampled from arterial or venous blood vessels. Thus, it is essential to test whether circulating miRNA profiles depend on the respective sampling site. We assessed potential differences in arterial and venous cell-free miRNA profiles in a cohort of 20 patients scheduled for cardiac surgery. Prior to surgery, blood was simultaneously sampled from the radial artery and the internal jugular vein. After precipitating crude EVs, we performed small RNA Sequencing, which failed to detect significantly regulated miRNAs using stringent filtering criteria for differential expression analysis. Filtering with less strict criteria, we detected four miRNAs slightly upregulated in arterial samples, one of which could be validated by reverse transcription real-time PCR. The applicability of these findings to purified arterial and venous EVs was subsequently tested in a subset of the initial study population. While an additional clean-up step using size-exclusion chromatography seemed to reduce overall miRNA yield compared to crude EV samples, no miRNAs with differential arteriovenous expression were detected. Unsupervised clustering approaches were unable to correctly classify samples drawn from arteries or veins based on miRNAs in either crude or purified preparations. Particle characterisation of crude preparations as well as characterisation of EV markers in purified EVs resulted in highly similar characteristics for arterial and venous samples. With the exception of specific pathologies (e.g. severe pulmonary disorders), arterial versus venous blood sampling should therefore not represent a likely confounder when studying differentially expressed circulating miRNAs. The use of either arterial or venous serum EV samples should result in highly similar data on miRNA expression profiles for the majority of biomarker studies. ACE inhibitors: Angiotensin-converting-enzyme inhibitors; ApoA1: Apolipoprotein A1; CNX: Calnexin; Cv: Coefficient of variation; cDNA: Complementary DNA; CABG: Coronary artery bypass graft; DGE: Differential gene expression; DPBS: Dulbecco's Phosphate Buffered Saline; EVs: Extracellular vesicles; log2FC: Log2 fold change; baseMean: Mean miRNA expression; miRNA: MicroRNA; NTA: Nanoparticle Tracking Analysis; NGS: Next-Generation Sequencing; RT-qPCR: Reverse transcription quantitative real-time PCR; rRNA: Ribosomal RNA; RT: Room temperature; SEC: Size-exclusion chromatography; snoRNA: Small nucleolar RNA; snRNA: Small nuclear RNA; small RNA-Seq: Small RNA Sequencing; SD: Standard deviation; tRNA: Transfer RNA; TEM: Transmission electron microscopy; UA: Uranyl acetate.