Cortisol levels in hair and fingernail samples could represent hormone levels that have accumulated over the past weeks and months. In this study, by using retrospective indexes, the associations between cortisol and the onset of acute coronary syndrome (ACS) were investigated among middle-aged and elderly men. We measured hair/fingernail cortisol levels in 73 ACS patients and 93 healthy controls; hair and fingernail samples for ACS patients were collected within a few weeks after the onset of ACS. The results indicated the patients exhibited significantly higher cortisol levels in their hair and fingernails compared with the healthy controls. In multivariate logistic regression analyses, adjusting for the traditional cardiovascular risk factors for ACS, high levels of hair or fingernail cortisol were associated with two- to three-fold increased risk of ACS, compared with low levels. We demonstrated that cortisol exposure over a relatively long period, assessed by hair and fingernail samples, was associated with the onset of ACS.