Assessing long-term cortisol secretion presents difficulties when cortisol measurement is carried out by saliva, plasma and urine analyses. Hair cortisol has gained increased interest as an alternative biological marker. So far, one study has been published studying hair cortisol in endurance athletes, showing higher levels compared to controls. Using accelerometer data in the present study, we cross-sectionally explored the relationship between moderate physical activity (MPA) and vigorous physical activity (VPA) levels and hair cortisol concentrations after taking into account age, gender, and perceived stress. Hair specimens were collected from 46 university students (20 males, 26 females, Mage ± SD =21.2 ± 1.87 years). Participants provided information about their socio-demographic background and levels of perceived stress. Accelerometer data were collected to assess physical activity. Cortisol concentrations were measured in the first 3-cm hair segment nearest to the scalp. MPA was not correlated with hair cortisol content (r = -0.08). A significant correlation was found between VPA and hair cortisol (r = 0.34, p < 0.05). A regression analysis revealed that participants with higher VPA had elevated hair cortisol concentrations even after taking into account age, gender and perceived stress (β = 0.33, p < 0.05, ΔR(2 )= 0.106). This is the first study showing that objectively assessed VPA is associated with increased hair cortisol levels in young adults. As VPA can be regarded as a physical stressor, it seems imperative that researchers consider participants' levels of VPA if they examine the relationship between stress exposure, hair cortisol and health.