Trier Social Stress Test (TSST)

Stress is one of the presumably most significant health problems of the twenty-first century (World Health Organization, 2001). Thus it has been of growing importance to gain insight into its underlying components via standardized methods, which reliably induce self-reported, behavioural, and biological stress responses in laboratory settings.
From a biological perspective two systems contribute to stress responses: The sympathetic-adrenal-medullary (SAM) axis and the hypothalamic-pituitary-adrenal (HPA) axis. Adequate methods are supposed to stimulate biomarkers for both systems, in order to investigate psychosocial stress and its implications. However often SAM activity, which supplies an organism with resources to perform typical “fight-or-flight” reactions, does not coincide with HPA activity. This is because endocrine stress responses seem to occur stressor-specific (Frankenhaeuser, Lundberg & Forsman, 1980; Lundberg, 1983), rather than unitarily, resulting in diverging and/or absent stress responses with the application of conventional stress-inducing methods like the cold pressure test, the Stroop test, and public speaking tasks. SAM activity is primarily elicited when an organism can influence the outcome of a challenging situation actively by effort, whereas for HPA activity effort in such a situation does not yield any positive feedback (Henry, 1992). Dickerson and Kemeny (2004) stated more precisely that effective HPA stressors need to incorporate mainly two components: (a) A social-evaluative threat and (b) uncontrollability of a situation an organism has to cope with.

With the development of the Trier Social Stress Test (TSST; Kirschbaum, Pirke & Hellhammer, 1993) a standardized protocol was introduced, integrating all above mentioned method criteria, and thus became a standard tool for the experimental induction of psychosocial stress in healthy adult as well as clinical samples. The TSST is a motivated performance task protocol being disguised as a job interview, which consists of a brief preparation period followed by two 5-minute test periods during which a subject has to perform a free speech and solve an arithmetic problem, respectively. Upon arrival the subject is informed by the experimenter via standardized instructions that he or her is supposed to take over the role of a job applicant who is invited for a personal interview with a selection committee. After ensuring that subject fully understood the instructions, he or her is guided into a separate room. The selection committee, which consists of a male and a female confederate of the experimenter being clothed in white lab coats, is specially trained to withhold any positive or negative feedback during the whole procedure and already seated on a desk upon entrance of the subject. Furthermore the room is equipped with a separate desk and chair, a video camera, and a microphone being located approximately 2 meters in front of the selection committee. After the initial 3-minute preparation phase, during which the subject has the opportunity to structure the upcoming free speech on personal job-relevant traits, the committee asks the subject to step in front of the microphone and begin with the presentation. Most test subjects finish their talk after about 2–3 min of speech time. Then the committee waits for approximately 20 seconds focussing the subject, and afterwards either requests to continue the speech since there was still time left, or begins to ask personal questions. After exactly 5 minutes the subject is asked to stop the speech and continue with the second task, which comprises continuous serial subtractions. The subject is told that upon each error, the committee would ask to start anew from the initial number. When in total 13 minutes have passed, the subject is asked to return to the experimenter who is already waiting in front of the room and continues with study protocol and the assessment of relevant biomarkers. 

With proper completion of the TSST about 70 - 85% (Kudielka, Hellhammer, & Kirschbaum, 2007) of all subjects reveal an increase in HPA activity as indicated by corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and serum and salivary cortisol (Kirschbaum et al., 1993). As compared to baseline, salivary cortisol levels gradually increase and usually peak with a two- to threefold magnitude within 20 minutes after stress cessation, while ACTH and serum cortisol peak immediately. Next to the stimulation of HPA markers, also SAM markers, e.g. salivary alpha amylase (cf. Rohleder et al., 2004), (nor-)epinephrine, heart rate responses, blood pressure, and electrodermal activity are increased by the TSST. In addition other endocrine and immune parameters like prolactin, growth hormone (GH), DHEA, and testosterone, as well as secretion of cytokines (Rohleder et al., 2001) are altered. Recently hemoconcentration as well as blood coangulation indicators were reported to change due to psychosocial stress (Kudielka et al., 2007). Evaluation of perceived stress level changes as measured with self-report scales also supports the validity of the protocol (e.g. Kudielka et al., 2004).
The methodological advantages of the TSST have led to its widespread use in psychoneuroendocrinological research (Kudielka et al., 2007), and stimulated the development of adaptions for children (TSST-C; Buske-Kirschbaum et al., 1997), retirees (Kudielka et al., 1998), psychiatric patients (Brenner et al., 2009) and groups (TSST-G; von Dawans, Kirschbaum, & Heinrichs, 2011). Furthermore a TSST-like placebo protocol has been developed, which is especially useful in studies employing control groups (Het et al., 2009). Apart from these, there have been plenty of other TSST variations, of which some resulted in altered biological stress responses (e.g. Gold et al., 2004; Gunnar et al., 2009; Kelly et al., 2007; Simoens et al., 2007). Thus it is suggested to stick with standard versions of the TSST in order minimize method variance and to achieve better comparability between different laboratories.

Certainly even with complete protocol standardisation there is considerable intra- and interindividual variation in biological stress response patterns. A well known confound for stress induced HPA activity are circadian secretion rhythms (Kudielka et al., 2004). This has to be considered when conducting the TSST by keeping the daytime of its performance constant and avoiding interference with the cortisol awakening response (cf. Pruessner et al., 1997). Endocrine reactions, which are due to preceding stressful events can also be eliminated by introducing a resting period of 30-45 minutes prior to TSST onset. However already anticipation and appraisal of the upcoming task can elicit a stress response (Gaab et al., 2005), which might confound interpretation of prestress HPA activity with HPA baseline activity. For within-subject experimental designs TSST applicability for repeated exposures is of potential interest. Prior knowledge about the protocol, as well as habituation to it alters HPA response to an interindividually varying extent (Wüst et al., 2005). Fortunately these effects of repeated TSST exposure can be bypassed quite well by changing setting variables, i.e. the selection committee and the test location. For the assessment of SAM activity with repeated TSST exposure such adaptions are not necessary, as its indicators fail to exhibit any habituation effect. The same seems to apply to cytokines, blood coangulation indices and parameters of hemoconcentration (Kudielka et al., 2007).
Further stress response variation can be explained by demographic, biological and psychological variables, which ought to be experimentally or statistically controlled. Among several variables, which exhibit influence on stress responses, e.g. age, uptake of nicotine (Kirschbaum, Scherer & Strasburger, 1994, Childs & de Wit, 2009), or caffeine (al’Absi et al., 1998), alcohol consumption (Zimmermann et al., 2004), dietary status (cf. Gonzalez-Bono et al., 2002; Epel et al., 2000), pregnancy (Nierop et al., 2006), lactation (Heinrichs et al., 2001), and personality, most consistently gender has been found to moderate salivary cortisol and ACTH secretion (Kudielka & Kirschbaum, 2005), i.e. men exhibit an output of 200% as compared to women. Thus variation in sex steroids levels might contribute to humoral responses to psychosocial stress. In line with this hypothesis women using estrogen-containing oral contraceptives show blunted salivary cortisol responses, although they exibit similar heart rate changes and perceived stress, as compared to women not using oral contraceptives (Kirschbaum, Pirke & Hellhammer, 1995). Additionally salivary cortisol and ACTH responses in women to the TSST seem to differ depending on their menstrual phase: Women in the luteal phase revealed salivary cortisol responses comparable to that of men, whereas in the follicular phase responses where similarly blunted as when using oral contraceptives (Kirschbaum et al., 1999). Corticosteroid binding globulin (CBG) level, which is known for being influenced by sex steroids and oral contraceptives, seems to be of major importance for the outlined issues (Kumsta et al., 2007). For more detailed information on potential confounds, Foley & Kirschbaum (2010) recently provided an extensive review on studies, investigating factors, which influence HPA responses to the TSST.



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