Cortisol is the primary glucocorticoid produced by the activation of the hypothalamic pituitary adrenal (HPA) axis after a psychological or physiological stressor. The dysregulation of the HPA axis by chronic stress has been associated with psychiatric disorders. Although hair is currently the main validated source of chronic cortisol concentrations, cortisol is also bound to human nails, another keratinised matrix. Therefore, nail cortisol has the potential to be an alternative retrospective chronic measure of HPA activation. The aim of this systematic review was to assess the methodological issues and functional correlates of nail cortisol. A total of 16 eligible human studies were extracted from Medline (PubMed and Ovid), ProQuest (PsycINFO), and Scopus. A qualitative synthesis of the literature found that immunoassays and mass spectrometry were the primarily methods of analysis. However, methodological variability remained evident between studies. Nail cortisol appeared to correlate with saliva and hair and was often measured in conjunction with dehydroepiandrosterone (DHEA). Lifetime stress events, but not perceived stress, were mainly associated with increased nail cortisol concentrations. It is possible that gender may have mediated these findings. Finally, higher levels of nail cortisol were associated with depression and acute coronary heart disease. In conclusion, nail cortisol may serve as a promising retrospective biomarker of chronic stress. However, very few studies have investigated the effects of covariates, such as age, gender, ethnicity, and nail characteristics. Despite promising evidence, further studies are required to support the utility of nail cortisol as a biomarker of chronic stress across the human lifespan.