Effect of Flavored Beverage Crystals on Salivary Cortisol Enzyme-Immunoreactive Assay Measurements M. Kathleen Gordon Elizabeth Peloso Ashley Auker Mary Dozier Department of Psychology University of Delaware Newark Delaware E-mail: mdozier@udel.edu ABSTRACT: We examined the effects of using flavored drink crystals as a salivary stimulant on salivary cortisol values. The effects of both amount and method of flavored crystal administration on cortisol values were evaluated using a high sensitivity enzyme immunoassay (EIA) kit to measure cortisol. Repeated saliva sampling, one sample without the drink crystal stimulant, followed immediately by a second sample using the drink crystal stimulant, allowed direct analysis of the effect of the stimulant on cortisol values. Repeated sampling, with the stimulant used when obtaining both the first and second samples, allowed analysis of the repeatability of cortisol measurements using the drink crystals. The results suggest that the use of drink mix crystals as an oral stimulant causes a small increase in measured cortisol values, as well as an increase in the variability of these values. The results also suggest that the effect of drink crystals is sufficiently regular so that it will not distort either within or between subject comparisons as long as there is consistency in use or non-use of the stimulant. ß 2005 Wiley Periodicals, Inc. Dev Psychobiol 47: 189–195, 2005. Keywords: salivary; stimulant; cortisol; enzyme immuno-assay Regulation of cortisol by children and infants is frequently studied because of the association of abnormal diurnal cortisol rhythms and stress reactivity with later psycho- pathologies and because of the association of early trauma with later abnormal cortisol levels in children (Bugental, Martorell, & Barraza, 2003; DeBellis et al., 1999; Gunnar & Vazquez, 2001). Salivary cortisol has become the standard method used to measure cortisol levels in children because sampling is non-invasive, repeatable, non-stressful to the subjects, and has been shown to correlate well with blood plasma cortisol measurements. However, interpretation of the results of cortisol assays across studies has been difficult because of both the variety of assay techniques and the variability in saliva sampling methodology used. Early salivary cortisol assays were performed using radioimmunoassay (RIA) kits designed for the measure- ment of cortisol in blood plasma (Schwartz, Granger, Susman, Gunnar, & Laird, 1998). The application of this methodology to test saliva required the use of relatively large saliva sample volumes because the amounts of cortisol in saliva are smaller than in blood. One of the challenges in measuring cortisol in infants and young children has been the difficulty of obtaining adequate saliva volumes to perform the assay (Luby et al., 2003). Agents to stimulate salivation, including chewable wax pledglets, sugar-free chewing gum, and placement of sweetened drink crystals in the mouth at the time of cortisol sampling have been used in children over 1 year of age (Bruce, Davis, & Gunnar, 2002; Doussard-Roosevelt, Montgomery, & Porges, 2003; Hardie, Moss, Vanyukov, Yao, & Kirillovac, 2002). Received 11 November 2004; Accepted 10 May 2005 Correspondence to: M. Dozier Contract grant sponsor: NIH Contract grant numbers: RO1MH52135, K02MH01782 Contract grant sponsor: NIH Contract grant number: R21MH065046 Contract grant sponsor: NIH Contract grant number: S2986091301 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/dev.20081 ß 2005 Wiley Periodicals, Inc.