Acute stress is increasingly recognized as a precipitant of acute myocardial infarction (AMI). However, the role of chronic stress in developing AMI is less clear. We have developed a method to measure cortisol in hair, which allows longitudinal assessment of cortisol levels prior to an acute event. We aimed to evaluate the hypothesis that chronic stress, as assessed by hair cortisol content, is associated with the development of AMI. A prospective case-control study included 56 patients admitted to hospital with AMI and 56 control patients, admitted to internal medicine wards for other indications. An enzyme immunoassay technique was used to measure cortisol in the most proximal 3 cm of hair, considered to represent the most recent 3 months of exposure. Median hair cortisol contents (range) were 295.3 (105.4-809.3)ng/g in AMI patients and 224.9 (76.58-949.9)ng/g in controls (p = 0.006, Mann-Whitney U-test). After controlling for other risk factors for AMI using multiple logistic regression, log-transformed hair cortisol content remained the strongest predictor (OR 17.4, 95% CI 2.15-140.5; p = 0.007). We demonstrated elevated hair cortisol concentrations in patients with AMI. This suggests that chronic stress, as assessed by increased hair cortisol in the 3 months prior to the event, may be a contributing factor for AMI.