Objective: In obese subjects a relatively high cortisol output in urine has been observed compared to nonobese individuals. However, cortisol levels in blood, saliva, and urine in association with obesity have been inconsistent across studies, possibly due to the high variability of systemic cortisol levels. Cortisol levels measured in scalp hair provide a marker for long-term cortisol exposure, and have been associated with cardiovascular disease in an elderly population and to disease course in Cushing's disease. We aimed to compare hair cortisol levels between obese patients and nonobese controls. Methods: Hair cortisol levels of 47 obese patients (median BMI 38.8, range 31.1-65.8), 41 overweight, and 87 normal-weight subjects using an enzyme-linked immunosorbent assay (ELISA) were measured. Results: Obese patients had higher hair cortisol levels than overweight and normal weight subjects (respectively 30.8 vs 8.5 and 8.4 pg/mg hair, P < 0.001). No significant difference in hair cortisol levels was found between normal weight and overweight subjects. Conclusions: Our results suggest a higher long-term cortisol exposure in obese patients, which may contribute to cardiovascular disease risk. Future research will determine whether long-term cortisol levels provide a novel treatment target in the management of cardiovascular disease risk in obesity.