Many space missions have shown that prolonged space flights may increase the risk of cardiovascular problems. Using a three-dimensional clinostat, we investigated human endothelial EA.hy926 cells up to 10 days under conditions of simulated microgravity to distinguish transient from long-term effects of 0g. Maximum expression of all selected genes occurred after 10 minutes of clinorotation. Gene expression (osteopontin, Fas, TGF-ß) declined to slightly upregulated levels or rose again (caspase-3) after the fourth day of clinorotation. Caspase-3, Bax and Bcl-2 protein content was enhanced for 10 days of microgravity. In addition, long-term accumulation of collagen type I and III and alterations of the cytoskeletal alpha- and beta-tubulins and F-actin were detectable. A significantly reduced release of soluble factors in simulated microgravity was measured for brain-derived neurotrophic factor, tissue factor, VEGF and interestingly for endothelin-1, which is important in keeping cardiovascular balances. The gene expression of endothelin-1 was suppressed under 0g conditions at day 7 and 10. Alterations of the vascular endothelium together with a decreased release of endothelin-1 may entail post-flight health hazards for astronauts.