Abstract Background Several studies have consistently shown that soluble CD40 ligand (sCD40L) concentrations are elevated in patients with acute coronary syndromes (ACS) and that it can be used as a biomarker for risk stratification. However, recently we could demonstrate that sampling techniques have impact on sCD40L measurements. Thus, it was the aim of our prospective study to evaluate the impact of sampling techniques on sCD40L concentrations of patients with acute coronary syndromes compared to controls. Methods and results We included a total of 60 patients, — 30 with an acute coronary syndrome, 10 with cardiovascular risk factors but no relevant coronary artery disease and 20 healthy individuals. Blood samples were collected in gel filled tubes without additives, EDTA filled tubes, or in citrate filled tubes. In EDTA or citrate plasma samples, sCD40L values were higher in patients with ACS compared to controls. In contrast, no difference in sCD40L values between ACS patients and controls was observed for serum samples. Conclusion Our data show that only plasma samples, but not serum samples are appropriate for sCD40L measurements. In general, preanalytic conditions are crucial for the assessment of sCD40L concentration and, thus, should be carefully considered for future studies.