The analysis of cortisol in human scalp hair is increasingly recognized as a promising new endocrine measure of long-term systemic cortisol secretion in the context of chronic stress research. Hair cortisol concentrations (hairF) are assumed to result from continuous incorporation of cortisol into slowly growing hair and to thus reflect integrated cortisol levels over the time period of hair growth. Due to their retrospective and long-term nature, hairF should be intra-individually stable and unaffected by situational variability. Over the past years, cumulative evidence has supported several of the general assumptions underlying hair cortisol analysis, such as the overall validity, high test-retest reliability of hairF as well as robustness to several potential sociodemographic or hair-related covariates. However, more research on fundamental methodological questions of this relatively new method is warranted. Importantly, the exact mechanisms of steroid hormone incorporation into hair are still not satisfactorily understood. External incorporation of cortisol, originating from sweat, is continually discussed as a potentially relevant source of hairF. Further, there are still concerns about whether hairF are indeed not altered by acute situational influences. To address these issues, one focus of the present research program was to explore the possibility that cortisol from sweat contributes to hairF and that hairF may be subject to acute sweat-inducing interventions. For this, the first part of this thesis includes the validation of an analytical procedure to measure cortisol concentrations in sweat (sweatF) and determination of sweatF in natural sweat samples (study I) as well as examinations on effects of incubating human scalp hair in a hydrocortisone-containing artificial sweat solution on hairF (studies II & III). Results show that cortisol is present in human sweat in concentrations that are strongly related to those in saliva. Further, the findings consistently reveal higher hairF after exposing hair to artificial sweat under in vitro and in vivo conditions, respectively. Combined, these findings show initial evidence for the assumption that cortisol in sweat may be a relevant source of hairF. In the second part of this research program, two independent experimental in vivo studies were conducted that explored whether hairF are subject to acute variation, potentially related to cortisol incorporation from sweat. For this, healthy individuals either underwent a treadmill run in which sweating together with a systemic cortisol reaction was provoked (study IV) or took part in a sauna bathing challenge in which sweat production was induced without systemic cortisol reactivity (study V). Findings show that hairF as well as hair cortisone concentrations (hairE) are not altered by single sweat-inducing interventions and unrelated to acute salivary cortisol reactivity. In addition, findings indicate that hairF and hairE were not subject to diurnal variation. Thus, the outcome of this work provides support for the ‘classical’ model of hair cortisol, which holds that hairF represent a stable retrospective marker of systemic cortisol production. Further, the findings substantiate the potential value of hairE for obtaining information on long-term systemic cortisol levels. Besides incorporation pathways, examinations on cortisol elimination mechanisms from hair are essential to understand the complex nature of hairF. Here, ultraviolet/sunlight radiation constitutes a potentially important factor that has received little attention in hair cortisol analysis so far. The third part of the present doctoral thesis thus conducted a first comprehensive investigation into this topic. Three studies examined the stability of cortisol molecules to radiant energy (study VI) and effects of a single short-term artificial light irradiation (study VII) and long-term naturalistic sunlight radiation (study VIII) on hairF and hairE in human scalp hair samples. Results consistently reveal that longer light exposure resulted in a decrease of hairF and hairE under in vitro conditions. However, the magnitude of this effect under real-life testing conditions needs to be determined in future research. Notwithstanding, our data strongly suggest that hair samples for steroid analyses need to be stored in a dark environment. In sum, overall findings of the current thesis provide insights into basic methodological aspects of hair cortisol analysis. The research proves the robustness of hairF (and hairE) to acute situational influences and thus further highlights the potential of hairF as an intra-individually stable measure primarily reflecting long-term systemic cortisol levels. However, the investigations also point to a possible confounding influence of individual perspiration on hairF, potentially related to external deposition of cortisol from sweat into hair. Further, the present thesis shows initial evidence for individual-level sunlight exposure as a possible confound in hair steroid analysis.