Research in urine proteomics and extracellular vesicles (EV) has gained increasing traction in biomarker research. Previous studies have suggested that both can be affected by urine protein concentration, however, there is a lack of data on the combined effect on urine proteomics. Here, we assessed the difference in proteomic signature in neat urine and urine enriched for EV at two protein concentrations, using two separate enrichment methods.