MacArthur SES & Health Network
MacArthur SES & Health Network

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Salivary Cortisol and Challenge Tests

Summary prepared by Clemens Kirschbaum and Dirk Hellhammer in collaboration with the Allostatic Load working group. Last revised October, 2000.

Chapter Contents

  1. Background — Challenge Tests
  2. Saliva Sampling and Storage for Free Cortisol Analysis
  3. Selected Readings on Saliva Collection and Steroids in Saliva
  4. Trier Social Stress Test
    1. General Protocol
    2. Tasks for the Different Experimenters
  5. Selected Readings on the TSST

Background — Challenge Tests

Challenge tests are very important means to assess reactivity and endogenous activity of the HPA axis, and salivary cortisol represents one of the easiest and most informative endpoints of HPA activity. Basal measurements of cortisol at wakening and in the evening provide an estimate of the diurnal rhythm, and responses to morning wakening and the Trier Social Stress Test provide estimates of reactivity that reflect ongoing life stress as well as the intrinsic potential of the HPA axis to respond.

Saliva Sampling and Storage for Free Cortisol Analysis

Several hormones, especially steroids, can be measured accurately and stress-free in saliva. Among these, cortisol has been used most often in the past 15 years of psychobiological and psychiatric studies of stress and stress-associated bodily complaints. Obtaining saliva samples is extremely easy and can generally be performed by the subjects/patients themselves.

Although there are several 'techniques' for saliva sampling, the easiest and most hygienic way is to collect saliva with the so called "Salivette" device (Sarstedt, Inc.). This device mainly consists of a small swab (which you might know from the dentist's) which the Ss gently chew on for 30-90 seconds until they feel that the swab is soaked with saliva. Note that there are three different Salivettes on the market: (a) the 'plain,' (b) the plastic-coated, and (c) the citric acid-stimulated Salivette. While (a) and (b) are usually well-suited for use with cortisol analysis, samples obtained with (c) may give false high values due to a low pH. To avoid a low pH with devices (a) and (b), Ss should not consume drinks with low pH immediately before saliva collection. If the citric acid stimulted devices are to be used, make sure your assay system is suited for handling low pH samples. If you want to assess other hormones or salivary immunoglobulin A (sIgA) from the same sample, use device (b). Some components of device (a) will cross react with some assays systems!

After having obtained the samples, they should be stored at -20 degrees C for hygenic reasons. However, since cortisol is a rather stable molecule, samples can be stored up to a minimum of 30 days at room temperature without a significant degradation of the steroid. If samples have to be shipped to a different laboratory for biochemical analysis, no dry ice or other form of refrigeration is necessary.

A PDF FAQs summary, Salivary Cortisol: Technical Issues written by Clemens Kirschbaum, is available.

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Selected Readings on Saliva Collection and Steroids in Saliva

Kirschbaum, C., & Hellhammer, D.H.(1989). Salivary cortisol in psychobiological research: an overview. Neuropsychobiology, 22, 150-169. [A PDF of this article is available.]

Kirschbaum, C., & Hellhammer, D.H.(1994). Salivary cortisol in psychoneuroendocrine research: recent developments and applications. Psychoneuroendocrinology, 19, 313-333. [A PDF of this article is available.]

Riad-Fahmy, D., Read, G.F., Walker, R.F., & Griffiths, K. (1982). Steroids in saliva for assessing endocrine function. Endocrine Reviews, 3, 367-395.

Trier Social Stress Test

The TSST is a procedure which was developed at the University of Trier for induction of moderate psychosocial stress under laboratory conditions with special emphasis on changes in HPA activity. In numerous studies in Trier and other laboratories, the TSST has proven to elicit significant changes in cardiovascular parameters, different endocrine axes as well as subjective stress ratings. To ensure that the results obtained in response to this challenge test remain comparable over time, it is necessary to standardize its execution, so that every subject (S) should be challenged similarly during confrontation with the TSST. Therefore, all persons involved in the TSST (experimenters involved in the committee, introduction & post-test assessments and debriefing; see below) should be familiar with the exact procedure of this stress protocol.

In addition to a general description of the test, specific duties of various experimenters are listed in detail below.

1) General Protocol

Each S spends about 60-70 minutes in the laboratory. Of that time, about 20 minutes are spent in the actual psychological stress protocol, the remaining time is spent on the introduction to the TSST, pre and post test assessments and debriefing.

On arrival the S is welcomed outside the experimental rooms by one experimenter responsible for S's reception and (later) introduction to the TSST. After a first instruction as well as questions regarding physical health, the S is led to experimental room #1, which serves as the rest and preparatory area. The S spends about 10 minutes there. This first resting phase is necessary to avoid potential activation of the hypothalamic-pituitary-adrenal axis (HPA), which could lead to the release of cortisol, and thus confound with later activation during the TSST. After ten minutes the S gives the first saliva sample.

The S is then led to experimental room #2. There, the S encounters the following experimental set-up: in the middle of the room is a microphone stand. At the other end of the room stands a video camera that is directed at the middle of the room with a table next to it. Behind the table sits a group of two or three people, who will be referred to as "the committee." On the table there is a cassette recorder that is connected to the microphone and a data protocol sheet in front of each committee member.

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The experimenter (who first greeted the S) explains to the S that he/she will have to deliver a speech for a job application to the committee, for which he/she would have ten minutes to prepare. This speech would be filmed and voice recorded. In addition, members of the committee would take notes regarding the manner and contents of the speech. He/ she should be informed that a member of the committee is trained in behavioral observation and that his/her behavior would be accordingly documented. As for the speech, the S should imagine that he/she applied for a position and was invited by that institution (Corporation, School, Department etc.) to introduce him/herself to the selection committee. The S is then informed that the speech should take about five minutes and that a second task would follow it, about which he/she would only learn more after delivering the speech. The experimenter then asks whether the S has any questions regarding the experimental protocol; any such questions will be directly answered.

Afterwards the S is led back to experimental room #1 where he/she has the opportunity to make written notes about his/her speech. The S is told that these notes may not be taken into the experimental room #2 but that they simply serve for the S's personal preparation. The S is also told that it is important to make a believable impression, because committee members will ask additional questions in case of disagreement. The S is then given ten minutes alone to ponder the contents of his/her speech. After ten minutes, an additional saliva sample is taken and the S is led back to experimental room #2. Upon entering the room, the video camera and cassette recorder are switched on by the chair of the committee. The S is asked to name his/her S number and to then start with his/her speech. All members of the committee remain quiet, as long as the S continues to speak fluently. Only after a pause of more than twenty seconds prior to the end of the five-minute period are questions asked, such as "What qualifies you in particular for this position?"

After the five-minute-period the S is then informed by the committee chair about the second half of his/her task, with an instruction such as: "We now want you solve a calculation task. Please count aloud backwards from 2083 to zero in 13-step sequences. Please calculate as quickly and correctly as possible. Should you miscalculate, we will point out your mistake and you have to start all over again. Do you have any questions?" This part of the test should be concluded after maximally five minutes, insofar as the S does not reach a count of "0" before that. The subject is then led to experimental room #3, where the post-test assessments and debriefing takes place. This consists of the collection of additional saliva samples and the filling out of questionaires. This part of the experiment will be concluded after maximally 30 minutes, so that the total duration of the experiment can be estimated to be about 60-70 minutes.

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2) Tasks for the Different Experimenters

2.1. 'Receptionist'

The experimenter (E) who receives the S is responsible for the instruction and timely coordination of the S. Because the exact schedule of the collection of saliva samples is essential in order to compare data, it is recommended that the E carries a stopwatch, if he/she should lack experience. When receiving the subject, it is important to ask about health-related limitations or possible stimulation of the HPA. Even though the S will be informed about participation conditions at the time of scheduling the appointment, this is still a good opportunity to double-check adherence to these conditions. One should ask at this point about illnesses, physical activities just prior to arrival, cigarettes smoked, and medications taken. In case of doubt: take notes and ask later; what is documented, can be controlled. Depending on the specific question of study, there are different exclusion criteria, of which the E should be informed; general exclusion criteria are, e.g. having a severe cold, recent physical exertion within the last 2 hours, larger meals, hormonal treatments. In addition, one could inquire at this point about study and career-goals (depending on the design of the study; in some experiments, such information is given as part of the subsequent presentation).

Before and during the rest period the S should not be informed in greater detail about the exact procedure of the experiment; if the S can exactly anticipate what comes his/her way, this in itself could have a stimulating effect on the HPA axis. If there should be any questions about the specific content of the experiment, the E should reply with a rather general statement about a psychological stress situation and refer to a later introduction for details. After the reception the S is led to the rest area; at this point the purpose of the rest can be explained to the S. Note the exact time of the beginning of the rest period.

After ten minutes, the S is brought from the preparatory room (#1) to the committee room (#2), where the exact protocol of the experiment is explained. Immediately before this explanation another saliva sample should be obtained (make sure to note the exact time again). A typical explanation of the experiment would be as follows: "Imagine you have applied for a job as a lawyer [or whatever the S's professional interest might be] and you were invited to present yourself before a committee which will evaluate on the basis of your personal characteristics. Your task in this experiment is to convince the committee in a free speech that you are the best candidate for the vacant position. Following these instructions, you have about ten minutes to prepare for the speech. You will later step in front of this line, so that your voice can be recorded by this microphone. Please also note that you will be recorded by a video camera as well. We will record your speech for a subsequent voice frequency analysis to reveal any paraverbal signs of stress. The camera recording is used for later behavioral analysis. The members of the committee are trained in behavioral analysis and will take notes during your speech. Following your speech, which is supposed to take five minutes, you will then be given a second task by the committee which will only be explained to you by the committee; that will also take about five minutes. Do you have any questions?"

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After this introduction the S is led back to the preparation room (#1), where he/she can prepare notes for his/her speech. Introduction and preparation together should take about ten minutes; depending on the introduction (usually about two minutes) there should be about eight +/- one minutes time for the S to prepare. The E should alert the S at this time to the fact that the written notes may not be taken into the speech room and that they only serve for the S's mental organization.

At the end of the preparatory period which should be spent by the S alone, another saliva sample should be taken; again note the exact time. The E then leads the S to the front of the door to the committee room (room #2); at this point final questions should be clarified, afterwards the E sends off the S to deliver his/her speech. With that, the job of the E is concluded. Usually one waits at this point for the next S's reception and introduction.

2.2. 'Committee'

The committee has contact twice with the S, during the introduction and during the actual stress phase. During the introduction the only duty of the committee is to be present. The S is supposed to get an impression who he/she will be dealing with later in the experiment. It is important to say something about the impression that the committee should make at this point. The principal aspect of the TSST is the role play, and for that it is important that all involved play their respective roles to the best of their abilities. As the committee which has to decide about the acceptance of an applicant for a specific position, the issue is therefore to make an impression that leaves no doubt about the seriousness of this endeavor. Furthermore, the TSST is meant to be a psychological stress situation; for that, as well, it is important to maintain a serious mood. In any case it should be avoided to talk about the situation as such. Any role play looses its realism (and with that its stress inducing effect), if it is made the subject of a discussion. It is clear that a real job interview would never take place like this in real life and that the TSST can only be a compromise—however, that should only, if at all, be discussed at the time of the introduction or post-test assessments and debriefing, but not during the speech task. Therefore it is recommended that during the introduction by the E, none of the committee members talks or laughs; should the S address the committee, one should only return the greeting courteously. If necessary, it can be pointed out that any questions of the S should be directed to the E, rather than the committee.The actual task of the committee starts when the S enters the room ten minutes later to deliver his/her speech. At that time the chairperson of the committee should turn on the videocamera by hand or remote control (make sure you know the operating instructions beforehand). He/she opens up the session with the words "Please step behind the line, name your S-number and begin your speech." Simultaneously the most left-seated committee member turns on the cassette recorder; although only a decoy, this gesture is of great importance in light of the situational context. Furthermore all committee members should seek eye contact with the S during the speech; the knowledge that all persons present give him/her their undivided attention further reinforces the seriousness of the situation for the S. Of course, there should be no laughing during the speech. Only the chairperson should address the S directly, so that coordination problems between the committee members can be avoided.

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One should let the S speak for the first three minutes. In most cases the S will come to the end of the speech even before three minutes have passed. One should give the S then time to formulate additional elaborations. In any case, there should be a pause. After about twenty seconds pause one can alert the S to the remaining time, as with the phrase "You still have time, please continue..." Should it appear after another ten seconds that the S has nothing further to say, then the chair should ask questions until the end of the time period. The phrasing of these questions is left to the chair's discretion; it may also be solely oriented on the S's previous statements. Typical questions in this context are:

  • Why do you think that you are the best applicant for this position?
  • What other experiences have you had in this area?
  • What about your studies identifies a special aptitude and motivation for this position?
  • Where else did you apply? Why?
  • What would you do, if your application here would not succeed?

Much has been said about psychological stress tests, therefore only a short remark at this point: the point of these questions is not to embarrass the S or be mean to him/her. That is neither the purpose nor the task of the TSST and would also distort the contents of this role play. The S's task is to present him/herself before an audience. The questions should serve to deepen this presentation and to receive information about specific qualities of the applicant. That's all. Questions such as "Do you have friends?" are in this context not called for.

In rare instances will the S be able to talk alone for the full five minutes. In that case it is left to the discretion of the chair whether he/she want to intervene between the third and fifth minute to ask questions to the S or whether the S is allowed to continue. This should also be dependent on what is being said by the subject. For instance, it is not appropriate for the applicant to speak in great detail about specific lessons one may have learned in the course of one's training at university or elsewhere. Some S's use their school-knowledge to distract from their own person. In that case the chair should certainly intervene, for example by saying: "We believe you that you know how to execute a market analysis, but we would be more interested to find out why you were so involved in or drawn to this area."

After the five minutes, it is the chair's duty to explain the second part of the stress protocol. To avoid annoyance of the S, it is very important to make it clear that this is indeed a second task that has nothing to do with the application speech. In the past, some S's refused to engage in mental arithmetic because they felt (rightly so) that it had nothing to do with their job application. A typical transition would sound like this: "Thank you very much, that should be enough for now. We now want to ask you to work on a second task. This one is about mental arithmetic. We ask you to count backwards to zero in 13-number steps, starting at 1687, and to do it as fast and correctly as possible. Should you miscalculate, you will be told so and you start again at 1687. Do you have any questions about this? ...Please begin, then."

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It is recommended to use prime numbers as subtractors for this task, because these make the job more difficult. As far as I know, only one S has ever been able to count backwards all the way to "0." Protocol sheets with all intermediate numbers are available as appendices in the committee room; the members of the committee do therefore not have to calculate themselves. Should the subject miscalculate, the chair will respond with the standard phrase "Error. 1687." until the end of the test period. Furthermore the committee should note the number of errors and the number that the subject eventually reached as a performance measure. At the end of the test period the chair should thank the S for his/her participation and ask him/her to go to the neighboring room for post-test assessments and debriefing. With that, the committee's role in the TSST is concluded.

2.3. 'Post-Test Assessments and Debriefing'

Usually only one E is performing post-test assessments and debriefing. The rules to be followed are relatively simple, but for the purpose of analysis and interpretation adherence they are important. Because the HPA axis only starts secreting cortisol about ten to fifteen minutes after the onset of the stressor, saliva samples will also be taken only after the end of the stress experience. Therefore it is very important to follow an exact timing of saliva or blood sampling. For this purpose, the E notes the exact time when the S enters the post-test assessments and debriefing room. Right as the S enters the room, he/she is asked to give a saliva sample. Additional samples should then be taken in ten-minute intervals. In between the S has an opportunity to fill out questionaires, insofar these are needed (depending on the experimental study). If there is only a single occurrence of the TSST in the course of the experiment, the E can give information about the purpose of the study, (i.e. "debrief" the S). This step, however, is not allowed, if additional runs through the TSST with this S should be planned. In that case the E can refer to later explanations about the experiment and the goals of the study. Following the last exposure to the TSST (in case the study protocol calls for repeated testing of the same subject), there should always be a detailed debriefing of the Ss which should include an explanation why the stress protocol had to be included for the current research question, a statement that no real audio or video recording or performance assessment was made etc. It is usually helpful, if the committee members participate in the debriefing ('look, in reality these people are not as bad as they had to be for this experiment').

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Suggestions regarding this protocol or further standardization of the planning and execution of the TSST are always welcome. Please direct your comments to:

Clemens Kirschbaum, Ph.D.
Institut für Physiologische Psychologie II
Universitätsstrasse 1
D-40225 Duesseldorf, Germany
Tel: (+49) 211-811-2090; -4384; fax: -2019
Cellular (+49) 170-286-7440

Selected Readings on the TSST

Kirschbaum, C., Pirke, K.-M., & Hellhammer, D.H. (1993). The 'Trier Social Stress Test'- a tool for investigating psychobiological stress responses in a laboratory setting. Neuropsychobiology, 28, 76-81.

Kirschbaum, C., Prüssner, J., Gaab, J., Schommer, N., Lintz, D., Stone, A.A., & Hellhammer, D.H. (1995). Persistent high cortisol responses to repeated psychological stress in a subpopulation of healthy men. Psychosomatic Medicine, 57, 468-474. [A PDF of this article is available.]

Kirschbaum, C., Schommer, N., Federenko, I., Gaab, J., Neumann, O., Oellers, M., Rohleder, N., Untied, A., Hanker, J., Pirke, K.-M., & Hellhammer, D.H. (1996). Short term estradiol treatment enhances pituitary-adrenal axis and sympathetic responses to psychosocial stress in healthy young men. Journal of Clinical Endocrinology and Metabolism, 81, 3639-3643.

Kirschbaum, C., Wüst, S., & Hellhammer, D.H. (1992). Consistent sex differences in cortisol responses to psychological stress. Psychosomatic Medicine, 54, 648-657.

Kirschbaum, C., Wüst, S., & Strasburger, C.J. (1992). 'Normal' cigarette smoking increases free cortisol in habitual smokers. Life Sciences, 50, 435-442.

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