Analysis was done using an electrochemiluminescence immunoassay (ECLIA) [26]. Samples of reference corticosteroid-users, as well as morning samples collected more than 5minutes too late/early and evening samples not collected between 7PM and 9PM were excluded. The detailed descriptive results were published elsewhere [27]. Hair cortisol The ChiBS study functions additionally as a pilot project evaluating the feasibility and validity of hair cortisol (and cortisone) measurements in healthy children and its applicability for large-scale psychosocial stress research in young children, as this is currently unexplored. Cortisol (and cortisone) concentrations were analyzed in hair samples obtained from the vertex posterior region of the scalp [28,29]. Sampling was done at school by trained researchers.

The hair samples were cut as close to the scalp as possible using clean, stainless steel scissors and tied together with a little cord to mark the proximal side. Only the most proximal 6cm of the hair strands were analyzed (approximately 150 to 200mg of hair), which is generally assumed to be the maximum length of hair to obtain a reliable estimate of systemic cortisol concentrations in the past (i.e. 6months) [29]. Hair samples were exclusively taken from girls to maximize the possibility that the hair reached the required length. If the length of hair was shorter than 6cm, no sample was taken. The hair samples were stored in a folded piece of paper in individual zip-lock bags in a dark, dry place and at constant temperature, to be analyzed 12months later at the University of Strasbourg in the Institute of Legal Medicine (Faculty of Medicine).

Hair cortisol has been shown to be stable over months to years, GSK-3 justifying the long time lag between sampling and analyzing. An amount of 50mg of hair was finely minced, after which cortisol was extracted from the hair samples. Cortisol concentrations were analyzed using a Liquid Chromatography-tandem Mass-Spectrometry technique (Acquity UPLC Waters, Quattro Premier XE and Mass Lynx software). The details of the methods and the validation parameters are described in a separate paper. Serum cortisol After an overnight fasting period, blood samples were obtained through venipuncture using local anesthesia with EMLA? patches (lidocaine+Prilocaine). Blood samples were centrifuged and serum was stored at -80��C. Serum cortisol was assayed using the same technique as salivary cortisol namely ECLIA. Heart rate variability HRV is defined as the variability of the time between consecutive R peaks of the electric QRST interval on the electrocardiogram. In a healthy situation, considerable variability reflects the heart��s ability to adequately respond to physiological and environmental stimuli.