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Psychosom Med. 2003 Mar-Apr;65(2):313-9.

Compliance with saliva sampling protocols: electronic monitoring reveals invalid cortisol daytime profiles in noncompliant subjects.

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Department of Experimental Psychology II, University of Düsseldorf, Germany.



Ambulatory saliva collections for subsequent analysis of free cortisol levels are now frequently applied to measure adrenocortical activity in healthy subjects and patient populations. Despite the prime importance of accurate timing of saliva collection outside the laboratory, no data are available on the compliance of study participants following a given sampling protocol. This study investigated how accurately subjects adhered to the instructions to collect six saliva samples throughout 1 day.


Subjects were instructed to collect six saliva samples throughout 1 day (directly after awakening, 30 minutes after awakening, 11 AM, 3 PM, 8 PM, 10 PM). Objective compliance was measured using an electronic monitoring device given to the subjects either with ("informed" N = 23) or without ("noninformed" N = 24) their knowledge of the nature of the device. Data on subjective compliance were obtained by self-report.


Thirty-one subjects (74%) were found to comply with the sampling instructions, and 11 (26%) failed at least once to obtain the saliva sample at the correct time of day. Nine of the 11 noncompliant subjects (82%) had two or more noncompliant samples. Fifty-five percent (6 of 11) of the noncompliant subjects took sample 2 outside the sampling window. The circadian cortisol profile differed significantly between compliant and noncompliant subjects (F = 7.98, p =.007). The most important effect of compliance was seen in the rise of cortisol at awakening. Compliant subjects showed a robust increase, whereas noncompliant individuals had only minimal changes from baseline at 30 minutes after awakening (t = 2.89, p =.007). Thus the steepness of the circadian cortisol decline was greater for compliant subjects (t = 2.10, p =.043). Furthermore, the informed group adhered more closely to the sampling protocol than the noninformed subjects (p =.001). Self-reported compliance also differed significantly between study groups (p =.03). In the noninformed group, self-reported sampling accuracy was significantly higher than objectively measured compliance (p =.03); the two measures were similar in the informed group (p = NS).


A significant number of subjects did not obtain saliva samples reliably in an ambulatory setting. This can partially invalidate the cortisol results and mask potential differences between subject groups of interest. We therefore recommend the use of electronic monitoring devices or other suitable methods and that study participants be informed about the device when ambulatory saliva collection is performed.

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