Hair cortisol concentrations (HCC) have been suggested to reflect long-term integrated cortisol levels, but most evidence of associations with co-variates is from small samples of healthy volunteers. The objective of this study was to describe the collection of hair samples in a large cohort study and report associations of demographic and health measures with HCC. We examined HCC measured from the 3cm hair segment near the scalp in 3507 participants (aged 59-83y) from The Whitehall II occupational cohort study of British civil servants. Hair samples were analysed using a column switching LC-APCI-MS/MS assay. Findings from mutually adjusted linear regression analyses revealed lower HCC in participants who reported use of hair dye [% difference (95%CI); -12.5 (-22.0, -1.9), p value=0.022] and evidence suggestive of differences by length of sample storage and seasonal variation. With regard to demographic variables, HCC was lower in women compared to men [-17.0 (-24.8, -8.4), p value