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Unlabelled: Cortisol is one of the most frequently used stress biomarkers in humans. Urine and saliva are the matrices of choice to longitudinally monitor cortisol levels. Salivary and urinary cortisol are often discussed as though they provide similar information. However, the relationship between "free" cortisol levels in urine (nonconjugated) and saliva (non-protein-bound) has yet to be properly evaluated using naturalistic designs., Objectives: To investigate the longitudinal relationship between salivary cortisol (SC) and first morning urinary cortisol (FMUC), and to compare the advantages and disadvantages of these matrices in assessing longitudinal changes in cortisol secretion using naturalistic designs., Methods: Cortisol levels from 31 healthy, Kakchiquel Mayan women in Guatemala were compared in one first morning urine (FMU) and four saliva specimens collected daily across three alternate days. Linear mixed-effect regression models including fixed and random effects were used to analyze the repeated-measures data., Results: FMUC levels (16.04-242.18 ng/ml) were higher than SC levels (0.21-5.16 ng/ml). A small but statistically significant relationship was found between FMUC and SC (each 1 ng/ml increase in FMUC predicted a 0.1% increase in SC; P < 0.05)., Conclusions: Nonconjugated FMUC levels are related to non-protein-bound SC levels collected throughout the day. FMU presents several advantages over saliva for the longitudinal assessment of cortisol in naturalistic studies. Cortisol levels are about 53-fold higher in FMU than in saliva, which makes between- and within-individual variation easier to detect, and FMUC levels are less likely to be affected by confounders than diurnal SC levels., (Copyright © 2013 Wiley Periodicals, Inc.)