Abstract Background/Introduction Chronic stress is considered to be an important element of residual cardiovascular risk and modern guidelines recommend its systematic assessment and treatment. Its pathophysiologic contribution, however, in acute myocardial infarction (AMI) was not, until recently, easy to elucidate. This arises from difficulties in objectively quantifying stress levels using conventional research methods, like questionnaires. Purpose The purpose of our study was to measure stress levels at the days preceding an acute myocardial infarction using a solid biomarker, Hair Cortisol Concentration (HCC). Methods 102 consecutive patients with AMI (71 patients with ST-elevation Myocardial Infarction [STEMI] and 31 with Non ST-elevation Myocardial Infarction) from our institution were recruited for this study. We took hair samples from them to precisely measure HCC, which reflects cumulative cortisol levels for the time period preceding the acute event. We also did full clinical, biochemical and psychological evaluation. HCC levels were then compared with 50 age-matched healthy controls. Results To assess the temporal trend of HCC in hair segments, corresponding to specific time periods, we fitted linear mixed models. The mixed effects linear model including the main effects of time and group (AMI versus controls) and their interaction showed that cortisol concentration in hair increased over time with higher rate of increase in AMI patients versus controls (beta of interaction = 3.57; 95%CI: 1.74, 5.40; p < 0.001) The model explained a high percentage of the variance (conditional R2=0.940). No other baseline variable had a significant effect when added to the basic model. There was no significant difference between STEMI and NSTEMI (beta of interaction = 1.15; 95%CI: -1.330, 3.59; p = 0.358) Conclusions Hair cortisol levels, presumably reflecting stress levels, were gradually increasing before an acute myocardial infarction with higher rate of increase in patients compared to healthy controls. There was no significant difference between STEMI and NSTEMI patients. Picture 1. Predicted hair concentrations of cortisol over time prior to the event, based on linear mixed models.