Hemodialysis (HD) patients are at high risk of cardiovascular disease and death. Reliable biomarkers for risk stratification and detection of acute myocardial infarction (AMI) are therefore pivotal. Cardiac troponins (cTn) are the preferred biomarkers for AMI. It remains unclear, if cTn concentrations changes as a consequence of HD treatment itself during dialysis. In this study, cTn was compared with soluble urokinase plasminogen activator receptor (suPAR) and Beta-2-microglobulin (B2M). We performed a prospective study including 17 HD patients measuring high-sensitive cardiac troponin t (hs-cTnT), suPAR and B2M before and after a dialysis session and verified the results in a random subgroup of eight patients from the group by repeating their measurements before and after a dialysis session 15 weeks later. Biomarker concentrations after dialysis were adjusted according to hemodilution or concentration according to the hemoglobin concentration. The average hs-cTnT concentration decreased significantly by −9.9% after dialysis (95% CI: −13.6% to −6.2%). The average (paired) difference were − 6.7 ng/L (p = 0.0104) after dialysis comparing 25 HD treatment occasions. SuPAR was not significantly influenced by dialysis. B2M decreased by −58% after HD as an expected result from the molecular size of the biomarker. The hs-cTnT in average decreased by −9.9% after dialysis. This is a diagnostic challenge since the current guidelines suggest a 20% change in hs-cTnT in patients with acute myocardial infarction. Larger prospective studies investigating the different factors influencing hs-cTnT after HD are warranted. Adjusting biomarker concentrations according to hemodilution or concentration using the hemoglobin concentration, should be considered in future studies to determine more exact changes in concentrations of cTnT and other relevant biomarkers.