Elaboration of the study question

Decide whether you are interested in acute cortisol responses or long-term cortisol elevations. Only use hair cortisol if you are interested in long-term cortisol elevations. Decide which function hair cortisol has in your study: independent, dependent, moderating, mediating or confounding variable. Reflect whether you expect positive or negative relationships; consider that both hyper- and hypocortisolism are conceivable. Decide whether your study question can be answered with cross-sectional data or whether longitudinal or experimental designs are necessary. Almost all prior studies were based on cross-sectional data. If you plan a longitudinal study, consider that you may not take hair from exactly the same vertex position for several months as the hair needs to grow back. Reflect whether and how the inclusion of hair cortisol can further your field of research. If you are interested in validity issues, describe why your study is important: Make sure that you provide information about the practical relevance and possible implications of your expected findings. If relating hair cortisol to stress, do not only assess levels of general perceived stress, but also gather information about objective or self-reported life events.

Role of vigorous exercise

Consider how and whether differing levels of vigorous exercise influence the study findings. Decide how and whether you will control for vigorous exercise (e.g. inclusion as a covariate, focus on inactive participants or athletes with equal training loads). Clearly define exercise intensity, i.e. light, moderate and vigorous exercise. Vigorous exercise is assumed to have a stronger influence on hair cortisol than light and moderate exercise. Decide whether to assess vigorous exercise via self-report or objective assessments. Consider not only the role of vigorous exercise, but also the one of physical fitness. The relative exercise intensity depends on the fitness level of a participant.

Instruction of participants

Elite athletes may expect data misuse: Provide adequate information about the purpose and the importance of your study and confirm in writing that the hair cortisol samples are only examined for scientific purposes. Inform participants that they should not cut their hair shorter than 3-6cm before the assessment (depending on the length of the retrospective period being studied). Ask participants not to colour their hair between baseline and post measurement in longitudinal or experimental studies.

Controlling sources of potential confound

Use multiple assessment tools that include medical history, questionnaires on perceived stress, life events, mental health or quality of life (to get information about different sources of stress). Assess information about participants’ social and demographic background (e.g. gender, age, race). Assess information about frequency of showering, contact with (chemically treated) water, and use of hair cortisol-altering products (e.g. shampoo, other hair products). Assess information about sauna use (heat) and sunlight exposure. Assess information about diseases related to altered cortisol secretion (i.e. Cushing’ s syndrome), pregnancy and use of cortisol-containing medication. Do not use more than two 3cm hair segments (representing the last 6 months). Ensure that the periods measured via hair cortisol correspond with those assessed via self-report measures.

Sampling

Ensure that your variables vary sufficiently: envisage using stratified sampling methods. Use power analysis to calculate the minimal/optimal sample size.

Data collection and hair cortisol analysis

Make sure that the study personnel is well-trained and with sufficient equipment. They must work quickly and be precise (e.g. all samples must be taken at the same vertex position, as near to the scalp as possible). Be aware of cultural issues. For instance, the religion of some female participants may not allow uncovering their head in front of a man. Make sure that enough hair is taken to analyze hair cortisol concentrations. Get in contact with the laboratory before you start data collection; inform the laboratory about the purposes of your study and make sure that the analysis procedures remain unchanged in longitudinal data assessments; discuss the total costs of the requested analyses.

Data analysis

Consider that relationships between hair cortisol and other variables may not be linear; consider using data analysis methods that allow examining dose-effect relationships. Check your data for univariate outliers; if you find outliers do not simply exclude them, but examine carefully why they have markedly increased hair cortisol concentrations. Explore whether your data is normally distributed. Otherwise, perform log-transformations before conducting statistical tests.

Data interpretation

Seek collaboration with and ask for advice by experts who have founded endocrinologic knowledge. Be careful when relating hair cortisol levels to health and disease; ensure that you do not over-interpret your findings. Clearly define your point of reference; whether you find hypocortisolism or hypercortisolism may depend on the reference group/norm you apply; compare the mean and range of your study with other investigations.