Aim Endothelial cells are exposed to different types of shear stress which very likely triggers the secretion of varying subsets of proteins. The aim of this study was to measure with sensitive proteomic techniques the secretome of endothelial cells under static, laminar and turbulent flow. Methods Freshly isolated human umbilical vein endothelial cells (HUVEC) were labelled with L- Lysine- ¹³C₆,¹⁵N₂ HCl and L-Arginine- ¹³C₆,¹⁵N₄ HCl for 5 cell doublings to differentiate between endogenously expressed proteins and serum proteins. Shear stress (laminar: 15 dyne/cm², turbulent: 8 dyne/cm², control) was applied for 24 hours using a cone-and-plate viscometer. Supernatant was then subjected to SDS-PAGE. The gel was divided into 20 samples, proteins were trypsinized in-gel and obtained peptides were analyzed using LC-MS (LTQ). Experiments were performed in duplicate. Results Under static control condition 395 proteins could be identified, of which 78 proteins (17%) were assigned to the secretome according to SwissProt database. Under laminar shear stress conditions 327 proteins (83 secreted, 22.6%) and under turbulent shear stress 507 proteins (79 secreted, 13.8%) were measured. We were able to identify 6 proteins specific for control conditions (e.g. bone morphogenetic protein 6, latent transforming growth factor beta-binding protein 3), 8 proteins specific for laminar shear stress (e.g. collagen alpha-1(XII) chain, latent transforming growth factor beta-binding protein 4) and 5 proteins specific for turbulent shearstress (e.g. endothelin-1, insulin-like growth factor II). Conclusion Using a cone-and-plate viscometer we were able to identify 587 proteins in the supernatant of HUVEC cultures from which only 101 proteins (17.2%) are known to be secreted. The identification of shear stress specific secreted proteins emphasizes the role of endothelial cells in modulating the plasma composition according to the physiological requirements.