Background: It is unknown whether the increased B-type natriuretic peptide (BNP) values found in ischemic heart disease are triggered directly by ischemia or whether they are caused indirectly by ischemia through diastolic contractures or regional wall motion abnormalities. Therefore, we investigated the BNP expression in isolated human muscle strips under conditions of ischemia with and without mechanical stress. Methods: Muscle strips (n = 90) were isolated from human right atria (n = 46). Contractures were induced by oxygen and glucose withdrawal. In 18 muscle strips contractures were prevented by means of butanedione monoxime (BDM). Sarcomere lengths were measured by electron microscopy (n = 12). The gene expression and protein amount of BNP were determined and compared to control muscle strips contracting under physiological conditions. Results: Hypoxia significantly decreased systolic force and induced diastolic contractures. This mechanical stress could be prevented in the group treated with BDM as evidenced by electron microscopy. Ischemia significantly increased BNP expression in both groups as evidenced by Northern blot analysis and immunohistochemistry. This increase was independent from mechanical stress. Conclusion: Our results indicate that ischemia is a potent mechanism for the expression of BNP. The increase in BNP expression under ischemic conditions is independent from concomitant mechanical alterations.