To inform the future use of hair cortisol measurement, we have investigated influences of potential confounding variables (natural hair colour, frequency of hair washes, age, sex, oral contraceptive (OC) use and smoking status) on hair cortisol levels. The main study sample comprised 360 participants (172 women) covering a wide range of ages (1-91 years; mean = 25.95). In addition, to more closely examine influences of natural hair colour and young age on hair cortisol levels, two additional samples comprising 69 participants with natural blond or dark brown hair (hair colour sample) as well as 28 young children and 34 adults (young age sample) were recruited. Results revealed a lack of an effect for natural hair colour, OC use, and smoking status on hair cortisol levels (all p's >0.10). No influence of frequency of hair washes was seen for proximal hair segments (p = 0.335) but for the third hair segment indicating lower cortisol content (p = 0.008). We found elevated hair cortisol levels in young children and older adults (p < 0.001). Finally, men showed higher hair cortisol levels than women (p = 0.002). The present data indicate that hair cortisol measurement provides a useful tool in stress-related psychobiological research when applied with the consideration of possible confounders including age and sex.