Why do we respond so differently? Reviewing
determinants of human salivary cortisol
responses to challenge
Brigitte M. Kudielkaa,b,*, D.H. Hellhammerb, Stefan Wu ¨stb,c
aJacobs Center on Lifelong Learning and Institutional Development, Jacobs University Bremen, Campus Ring 1,
28759 Bremen, Germany
bPsychology, Department of Clinical and Physiological Psychology, University of Trier, Johanniterufer 15, 54290 Trier, Germany
cDepartment of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, J5, 68159 Mannheim, Germany
Received 16 July 2008; received in revised form 1 October 2008; accepted 8 October 2008
Psychoneuroendocrinology (2009) 34, 2—18
Trier Social Stress Test
elucidating the biological pathways linking stress and disease is of substantial importance.
However, the identification of mechanisms underlying a dysregulation of major components of
the stress response system is, particularly in humans, a very challenging task. Salivary cortisol
responses to diverse acute challenge paradigms show large intra- and interindividual variability.
In order to uncover mechanisms mediating stress-related disorders and to potentially develop
new therapeutic strategies, an extensive phenotyping of HPA axis stress responses is essential.
Such a research agenda depends on substantial knowledge of moderating and intervening
variables that affect cortisol responses to different stressors and stimuli. The aim of this report
is, therefore, to provide a comprehensive summary of important determinants of, in particular,
human salivary cortisol responses to different kinds of laboratory stimuli including acute
psychosocial stress as well as pharmacological provocation procedures. This overview demon-
strates the role of age and gender, endogenous and exogenous sex steroid levels, pregnancy,
lactation and breast-feeding, smoking, coffee and alcohol consumption as well as dietary energy
supply in salivary cortisol responses to acute stress. Furthermore, it briefly summarizes current
knowledge of the role of genetic factors and methodological issues in terms of habituation to
repeated psychosocial stress exposures and time of testing as well as psychological factors, that
have been shown to be associated with salivary cortisol responses like early life experiences,
social factors, psychological interventions, personality as well as acute subjective-psychological
stress responses and finally states of chronic stress and psychopathology.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determinants of salivary cortisol responses to challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Gender and sex steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1. Gender, endogenuous sex steroids, menstrual cycle phase, oral contaceptives and
corticosteroid binding globulin (CBG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2. Sex steroid supplementation and hormonal replacement therapy . . . . . . . . . . . . . . . . . . . . . .
2.3.Pregnancy, lactation and breast-feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Nicotine, coffee, alcohol and dietary energy supplies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.Genetic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6. Methodological factors: habituation and time of testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7.Early life experiences: pre- and postnatal stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.8. Social support, social hierarchy and psychological interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.9.Personality factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.10. Acute subjective-psychological stress responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.11. Chronic stress, burnout and vital exhaustion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.12. Clinical studies and medication intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Final remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
is the large variation in the response magnitude between
individuals as wellas across different situations ortests. Such
variability can be observed with respect to the net cortisol
output as well as the time course of hormone secretion after
stress. Overall, the identification of mechanisms that deter-
mine the regulation and especially dysregulation of free
cortisol responses to stress is, particularly in humans, a very
challenging task. Since stress and stress-related health
impairments have become major problems in human life,
investigations into the biological pathways linking stress and
disease are of major importance. An extensive phenotyping
including salivary cortisol responsivity is essential in order to
be able to uncover mechanisms mediating stress-related
disorders and to potentially develop new therapeutic stra-
tegies in the future. Such a research agenda depends on
substantial knowledge of moderating and intervening vari-
ables that affect free cortisol responses to different kinds of
stressors and stimuli.
Besides naturally occurring acute and chronic stressors,
acute cortisol responses can be stimulated in the laboratory
by very different means including psychological stress pro-
tocols (e.g., cognitive tasks or public speaking paradigms like
the Trier Social Stress Test (TSST)), a wide variety of phar-
macological stimulants (e.g., administration of CRH, vaso-
pressin or synthetic ACTH, etc.), intense physical exercise or
even intake of standardized meals. Generally, for a research
setting, laboratory stress protocols offer the advantage of
standardization across test sessions but might lack the eco-
logical validity of field studies or ambulatory assessments.
Laboratory psychological stress tasks have different
potencies in their ability to reliably evoke salivary cortisol
responses (Biondi and Picardi, 1999). In a meta-analysis
covering 208 laboratory stress studies, Dickerson and Kemeny
(2004) investigated conditions capable of eliciting HPA axis
stress responses. They concluded that motivated perfor-
mance tasks reliably elicited ACTH and cortisol responses
if they were uncontrollable or characterized by social-eva-
luative threat. Tasks containing both elements were asso-
ciated with the largest hormonal changes and the longest
recovery times. More than 15 years ago, a psychosocial stress
task was developed, which is characterized by both
uncontrollable and social-evaluative elements. As it was
developed in Trier, it was eventually named the Trier Social
Stress Test. The TSST is a brief and highly standardized
laboratory stress task consisting of a preparation period, a
free speech and mental arithmetic task in front of an audi-
ence (Kirschbaum et al., 1993a) repeatedly showing cortisol
responder rates of over 70% (Kudielka et al., 2007c,d).
While psychological stressors are central stimuli that
require processing at higher brain levels, pharmacological
challenges act at different levels of the HPA system and
operate in a dose-dependent manner. For example, when
assessing adrenal cortex functioning via release of cortisol
from the adrenal cortex with an administration of a small
dose of synthetic ACTH (e.g., 1 mg Synacthen) one would test
for adrenal cortex sensitivity while administration of a larger
dose (e.g., 250 mg Synacthen) would assess its maximum
capacity (Daidoh et al., 1995). Many HPA axis stimulation
tests trigger increases in cortisol via pharmacological stimu-
lants acting primarily at the pituitary level, like exogenously
administered CRH or vasopressin. When interpreting and
comparing pharmacological provocation tests, researchers
should be aware of the fact that study results are largely
dependent on the applied stimulant and the chosen dosage.
Generally, researchers applying pharmacological stimulants
use very heterogeneous study approaches and designs and
therefore yield very diverse results. For instance, in humans
exogenously administered human CRH (hCRH) binds with high
affinity to endogenous CRH-binding proteins which show low
Determinants of salivary cortisol responses to challenge3
affinity to ovine CRH (oCRH) (Sutton et al., 1995). Conse-
quently, this results in very differential pharmacological
effects. In the pharmacological testing of HPA axis regula-
tion, reported outcome variables are typically ACTH and
total plasma cortisol levels. Salivary cortisol concentrations
are less frequently measured although the amount of salivary
cortisol predominantly reflects the free, biologically active
high correlations with total cortisol levels (Vining et al.,
1983; Kirschbaum and Hellhammer, 1989, 1994, 2007); note:
compared to blood, absolute levels of cortisol are lower in
saliva due to a relative abundance of the cortisol-metaboliz-
ing enzyme 11b-hydroxysteroid dehydrogenase type 2 (11b-
HSD-2) converting active cortisol into inactive cortisone
(Smith et al., 1996; van Uum et al., 2002). Further questions
regarding analytical laboratory procedures are discussed by
Gierens et al. (under revision).
Beside psychological stress tasks and pharmacological
stimulation, intense physical exercise can elicit significant
cortisol responses. Maximal exercise reaching the level of
exhaustion leads to significant hormonal increases as well as
sustained physical load exceeding 70% of the maximum oxy-
gen uptake (VO2max) (Luger et al., 1987; O’Connor and
Corrigan, 1987; Kraemer et al., 1989; Wittert et al., 1991;
Kirschbaum et al., 1992c). In contrast, short-term physical
exercise with a lower workload appears to exert no or only
minor cortisol responses (Friedmann and Kindermann, 1989;
Kirschbaum et al., 1993b, 1994; Lovallo et al., 2006). How-
ever, significant plasma cortisol responses can be observed
for hours (Deuster et al., 1992). In contrast to cortisol
responses to psychological stress (see below), there seems
to be no pronounced habituation effect in exercise-induced
cortisol responses (O’Connor and Corrigan, 1987).
Finally, researchers should be aware of potential meal-
et al., 2006). Proteins have been primarily discussed as
cortisol-stimulating agents (Slag et al., 1981; Anderson
et al., 1987; Gibson et al., 1999; Benedict et al., 2005).
Interestingly, standardized meals affect at least plasma
cortisol levels differently according to time of day with higher
meal-related increases at lunchtime (Brandenberger et al.,
evening (Quigley and Yen, 1979; Follenius et al., 1982).
2. Determinants of salivary cortisol
responses to challenge
The aim of this report is to provide an overview of important
determinants of salivary cortisol responses to stress in
humans demonstrating the role of age and gender, endogen-
ous and exogenous sex steroid levels (e.g., the female
menstrual cycle, use of oral contraceptives and hormone
ing, smoking, coffee and alcohol consumption as well as
dietary energy supply in salivary cortisol responses to acute
stress. Furthermore, we briefly summarize current know-
ledge of the role of genetic factors and methodological issues
in terms of habituation to repeated psychosocial stress
exposures and time of testing as well as psychological factors
that have been shown to be associated with salivary cortisol
responses like early life experiences, social support and
social hierarchy, psychological interventions, personality
factors as well as acute subjective-psychological stress
responses and finally states of chronic stress and psycho-
pathology. In view of the large number of potential sources of
inter- and intraindividual response differences and the
various laboratory challenge paradigms, we here necessarily
review a selection of studies on human salivary cortisol
responses to different types of challenges. Based on the
available research on salivary cortisol, we mainly focus on
responses to psychosocial laboratory stressors.
In the last decades, several animal and human studies inves-
tigated whetheragingaffects thefunctioningoftheHPA axis,
for example, by exploring if age is related to a reduced
resilience or less flexible functioning of this hormonal system
(for a review and meta-analysis see Seeman and Robbins,
1994; Otte et al., 2005).
Surprisingly, still only very few studies are available
reporting on salivary cortisol responses to psychological
stress tasks. Nicolson et al. (1997) did not observe any age
effects using a laboratory speech task, though task-related
increases were very low. In accordance, we did not observe
any age effects in salivary cortisol responses to psychosocial
et al., 1999). In men, we found no or only marginally higher
responses in the older subjects (Kudielka et al., 2000; Roh-
leder et al., 2002). In a reanalysis of five independent studies
from our laboratory with a total of 102 children, younger as
well as older adults who were exposed to the Trier Social
Stress Test (Kudielka et al., 2004a), we found elevated
salivary cortisol responses in the group of elderly men (for
sex difference see also below). Further very recent develop-
mental studies indicate that salivary cortisol responses to
psychological stressors in children and adolescents show
age-related changes as discussed in another chapter of this
issue (see Gunnar and Talge, in press).
In respect to pharmacological stimulation of the HPA axis
with CRH with or without dexamethasone premedication or
administration of CRH combined with vasopressin, the major-
ity of studies showed elevated HPA axis responses in elderly
et al., 1991; Waltman et al., 1991; Greenspan et al., 1993;
Heuser et al., 1994b; Born et al., 1995; Luisi et al., 1998;
Vgontzas et al., 2001), including salivary cortisol responses
(Kudielka et al., 1999). By administration of the 11b-hydro-
xylase-inhibitor metyrapone, the final enzymatic conversion
into cortisol from its precursor molecule 11-deoxycortisol can
be blocked in the adrenal cortex. Studies by Wilkinson et al.
and mineralocorticoid feedback-sensitivity in older age after
metyrapone treatment combined with a subsequent infusion
of exogenous cortisol or the synthetic mineralocorticoid flu-
drocortisone. Direct stimulation of the adrenal cortex with
synthetic or extracted ACTH does not point to a generally
altered adrenal cortex capacity or sensitivity with progressive
age (Kley et al., 1975; Parker et al., 1981, 2000; Vermeulen
et al., 1982; Ohashi et al., 1986b; Roberts et al., 1990;
Rasmuson et al., 1998; Martinez-Taboada et al., 2002). Only
4B.M. Kudielka et al.
in a very low dose ACTH-test (0.06 mg), Giordano et al. (2001)
could observe a reduced adrenal cortex sensitivity in elderly
After physical exercise,
unchanged or showed, in the main, comparable increases
in younger and older men as well as women (Ha ¨kkinen and
Pakarinen, 1995; Ha ¨kkinen et al.,1998; Kraemer et al.,1998,
1999; Traustadottir et al., 2004). However, these studies only
Age-related differences in HPA axis functioning are often
explained by assumptions derived from the so-called gluco-
corticoid cascade hypothesis introduced by Sapolsky et al.
(1986). This intriguing model, which was derived from
numerous animal studies, proposes that age-related altera-
tions in HPA axis regulation emerge due to a decrease in the
ability of hippocampal neurons to maintain a sufficient nega-
tive feedback function. In view of contradicting evidence, De
tor balance theory, proposing that even with older age
homeostatic control could be maintained by a new balance
between glucocorticoid and mineralocorticoid receptors,
resulting in similar endocrine responses to stress in young
and old subjects.
2.2. Gender and sex steroids
2.2.1. Gender, endogenuous sex steroids, menstrual
cycle phase, oral contaceptives and corticosteroid
binding globulin (CBG)
One of the most consistent findings employing psychological
tal harassment) is the significantly larger salivary cortisol
response in healthy adult men compared to women following
short-term laboratory stress (Steptoe et al., 1996; Nicolson
et al., 1997; Earle et al., 1999; Seeman et al., 2001; Lovallo
different, we also repeatedly demonstrated that salivary
cortisol responses to psychosocial stress differ between males
and females applying the TSST (Kirschbaum et al., 1992c,
1995b; Kumsta et al., 2007b). Interestingly, salivary cortisol
increases in men are up to twice as high as in women. The
typical mean response magnitude in men ranges from 200 to
400% increase from baseline whereas in women 50—150%
changes are usually found. Moreover, in men the sole antici-
pation of an upcoming psychosocial stress task led to a signi-
ficant saliva cortisol response even when they were not
actually confronted with the stressor. A similar anticipatory
endocrine response was absent in women (Kirschbaum et al.,
1992b). Meanwhile, we observed this sex difference in adre-
nocortical responsivity in more than a dozen studies (for
reviews and meta-analysis see Kudielka and Kirschbaum,
2005; Otte et al., 2005; Kajantie and Phillips, 2006; Kudielka
et al., 2007b).
In respect to the female menstrual cycle phase earlier
studies only reported on plasma cortisol responses (Abpla-
nalp et al., 1977; Tersman et al., 1991). In a follow-up study
on sex differences in HPA axis responses to psychosocial
stress, we investigated the role of the menstrual cycle
phase applying the TSST in 81 men, women in the follicular
phase of the menstrual cycle, women in the luteal phase,
and women using oral contraceptives (Kirschbaum et al.,
1999). While no sex differences emerged for total plasma
cortisol, salivary cortisol responses differed significantly
between groups. Women in the luteal phase had saliva
cortisol stress responses comparable to those of men
whereas women in the follicular phase or women taking
oral contraceptives showed significantly lower salivary
cortisol responses. Other studies replicated the findings
of comparably high salivary cortisol stress responses in
men and women during the luteal phase (Rohleder et al.,
2001; Wolf et al., 2001) as well as higher salivary cortisol
responses in women during the luteal phase versus women
taking oral contraceptives (Rohleder et al., 2003). Such
results underline the importance of strictly distinguishing
between the total cortisol secretion and the levels of
bioavailable free cortisol, as can be measured in saliva.
These and earlier studies that also described blunted sali-
vary cortisol stress responses in women taking oral contra-
ceptives (Kirschbaum et al., 1995b, 1996a) raised the idea
of a possibly modulating role of corticosteroid binding
globulin since steroid binding globulin levels in the blood
(including CBG) are significantly altered by intake of oral
contraceptives containing the synthetic estradiol compo-
nent ethinylestradiol (Moore et al., 1978; Wiegratz et al.,
2003). Indeed, Kumsta et al. (2007a) could recently report
on a significant negative correlation between CBG and
salivary cortisol levels after TSST exposure in 115 women
taking oral contraceptives while this correlation was not
observed in the 93 medication-free men.
In respect to age, the same sex effect emerged for elderly
subjects with men evincing higher salivary cortisol responses
(Steptoe et al., 1996; Nicolson et al., 1997; Kudielka et al.,
1998, 2004a). Although, we and others did not observe this
sex effect in saliva cortisol responses in children after TSST
exposure (Kudielka et al., 2004a) or in newborns undergoing
discharge examinations (Gunnar et al., 1989), studies in
newborns suggest that sex differences might be already
present at birth. Male neonates had higher saliva cortisol
responses when exposed to the standardized Brazelton neo-
natal behavior assessment (Davis and Emory, 1995), but girls
showed a higher salivary cortisol response to heel prick as
part of a standard pediatric examination (Buske-Kirschbaum
et al., 2004). Unfortunately, several other stress protocols
failed to elicit significant salivary cortisol increases in boys
and girls (Hardie et al., 2002). It should finally be noted that
other studies on heel stick blood draws, discharge examina-
tions or infant inoculations also did not find sex differences in
total plasma cortisol responses, supporting the finding that
sex differences can not be reliably found in childhood (see
Gunnar and Talge, in press).
Nevertheless, the possibility that different psychological
stress protocols cause stressor-specific salivary cortisol
responses should be acknowledged since some studies point
to the potential importance of the gender relevance of
psychological stressors. For example, Stroud et al. (2002)
reported that men but not women had significant saliva
cortisol increases after confrontation with an achievement
challenge (mathematical and verbal tasks) whereas women
but not men showed significant salivary cortisol responses to
a social rejection challenge. Though, in the meta-analysis of
Dickerson and Kemeny (2004) the importance of the two
identified key stress-producing factors, namely uncontroll-
ability and social evaluative-threat, was not modified by the
sex of study participants.
Determinants of salivary cortisol responses to challenge5
Regarding pharmacological stimulation, CRH administra-
tion with and without dexamethasone premedication repeat-
edly resulted in higher total plasma cortisol responses in
women compared to men (Greenspan et al., 1993; Heuser
et al., 1994b; Born et al., 1995; Luisi et al., 1998; Ku ¨nzel
et al., 2003), though Kirschbaum et al. (1992c) did not
observe any sex differences in a study measuring salivary
cortisol responses after hCRH stimulation. A change in endo-
genous sex steroid levels due to ovariectomy in premeno-
pausal women resulted in a slightly earlier but unchanged
plasma cortisol peak after CRH injection (De Leo et al., 1998)
while others reported on heightened basal urinary free
cortisol levels and simultaneously reduced plasma cortisol
responses to oCRH in estrogen-deficient young women with
clinical amenorrhea (Biller et al., 1990). Salivary cortisol
levels have not been reported in such studies. Likewise, in
102 healthy children, a maximal stimulation of the adrenal
cortex with 250 mg synthetic ACTH did not render significant
sex differences in total plasma cortisol responses across five
different age cohorts (Lashansky et al., 1991). In the earlier
mentioned study investigating the impact of sex, the
female menstrual cycle phase and use of oral contraceptives
(Kirschbaum et al., 1999), we also tested adrenal cortex
capacity by injecting 250 mg synthetic ACTH. While no group
differences emerged for total plasma cortisol, significant
differences emerged for the salivary cortisol response to
Synacthen. Women in the luteal phase of the menstrual cycle
had the highest salivary cortisol responses followed by men,
women in the follicular phase and users of oral contracep-
In response to physical exercise, the majority of studies
did not reveal any sex differences in salivary as well as total
plasma cortisol (Friedmann and Kindermann, 1989; Kraemer
et al., 1989; Kirschbaum et al., 1992c). Finally, after a
standardized meal, Lovallo et al. (2006) reported on higher
saliva cortisol increases in women compared to men.
The described sex differences in HPA axis responses to
stress may be due to sexual dimorphisms in brain structure
and function (Patchev et al., 1995; Rhodes and Rubin, 1999;
Cahill et al., 2001; Killgore and Yurgelun-Todd, 2001; Shors
et al., 2001; Wang et al., 2007). Beside the impact of
circulating CBG levels (Kirschbaum et al., 1999; Kumsta
et al., 2007a), further prime candidates for explaining such
observations are differences in the secretion of central
arginine vasopressin (AVP) levels or circulating gonadal ster-
oids with their complex effects on glucocorticoid and miner-
alocorticoid receptor regulation and functioning across men
andwomen (forreviews anda meta-analysis seeKudielka and
Kirschbaum, 2005; Otte et al., 2005; Kajantie and Phillips,
2006; Kudielka et al., 2007b).
2.2.2. Sex steroid supplementation and hormonal
Whereas many animal studies directly investigated the
impact of estrogens on HPA axis regulation, only few experi-
mental humans studies have been conducted with extremely
rare reports on salivary cortisol responses.
In a placebo-controlled study, an 8-week estradiol sup-
plementation in premenopausal women resulted in reduced
plasma cortisol responses to mental arithmetic (Komesaroff
et al., 1999) and Lindheim et al. (1992) reported a stress-
induced HPAaxis response before estradiol treatment but not
after a 6-week sex hormone replacement in postmenopausal
is merely based on habituation effects to the repeatedly
applied stress procedure. Del Rio et al. (1998) could not
show any estradiol effects on HPA axis responses in a
cross-over design, applying a relatively mild stressor. This
result is in line with findings in an own study measuring
salivary cortisol responses to stress. A 2-week estradiol
treatment in postmenopausal women did not alter TSST-
induced salivary cortisol responses, but HPA axis feedback
regulation as measured by the combined Dex-CRH-test was
increasedin the estradiol-substituted
women (Kudielka et al., 1999). Finally, Slayden et al.
(1998) did not observe any changes in plasma cortisol
responses before and after a 3-month treatment with estra-
diol in postmenopausal women applying an infusion with
continuously increasing levels of synthetic ACTH after dex-
amethasone premedication. From other studies no clear
conclusions can be drawn due to small sample sizes and
methodological problems (Collins et al., 1982; Liu et al.,
1987; Burleson et al., 1998).
In contrast, in healthy young men, the application of an
estradiol-containing patch for 48 h resulted in elevated sali-
vary cortisol responses to the TSST (Kirschbaum et al.,
1996b). However, in estrogen supplemented hypogonadal
men with a diagnosis of cancer, Komesaroff et al. (2002)
reported attenuated total plasma cortisol responses to
psychological stress while in male cancer patients treated
with synthetic estrogens for at least 6 months no changes in
plasma cortisol levels after dexamethasone treatment or in
response to synthetic ACTH could be found (Schu ¨rmeyer
et al., 1986).
Finally, to elucidate the impact of the sex hormone pre-
cursor dehydroepiandrosterone (DHEA), we conducted a pla-
cebo-controlled double blind study investigating HPA axis
stress responses to the TSST in 75 men and women of
advanced age after a 2-week DHEA or placebo treatment
(Kudielka et al., 1998). While no differences emerged for
salivary cortisol responses between the DHEA versus placebo
treated group, DHEA-treated women showed ACTH stress
responses similar to those of men but significantly enhanced
compared to women after placebo treatment.
Data on the impact of androgens and progestins on HPA
axis regulation in humans are extremely sparse and available
results so far do not suggest a significant mediation of stress-
related plasmaorsalivary cortisolresponses (Lindheimetal.,
1994; Burleson et al., 1998; Rohleder et al., 2002).
To conclude, it is obvious that the available data on the
impact of sex steroid supplementation or hormonal replace-
ment therapy in humans is very heterogeneous. Especially,
studies applying a potent challenge to elicit cortisol
responses in an experimental setting are still extremely
scarce. Based on the existing evidence, we can at least
conclude that even short-term sex steroid administration
may modulate acute HPA axis stress responses as well as
HPA axis feedback functioning.
2.3. Pregnancy, lactation and breast-feeding
Generally, pregnancy is accompanied by significant changes
in HPA axis physiology characterized by marked increases in
CRH, ACTH, plasma cortisol and CBG levels as well as
6 B.M. Kudielka et al.
somewhat elevated free cortisol levels (McLean et al.,
1995; Mastorakos and Ilias, 2003; de Weerth and Buitelaar,
2005; Lindsay and Nieman, 2005). Interestingly, salivary
cortisol responses to the cold pressor test or hCRH chal-
lenge during pregnancy were found to be dampened
(Schulte et al., 1990; Kammerer et al., 2002; for review
see de Weerth and Buitelaar, 2005). It was hypothesized
that this might be due to high circulating glucocorticoid or
CRH levels since high concentrations could act by feedback
mechanisms to blunt HPA axis responses to challenge, a
desensitization of corticotrophic cells in the pituitary or, at
least in part, the presence of CRH-binding proteins in
maternal plasma that reduce the concentration of circulat-
ing potentially bioactive CRH. In animals, lactation has
been associated with attenuated hormonal responses to
different kinds of stressors (Carter and Altemus, 1997). It
was therefore assumed that the human endocrine stress
response might also be moderated by lactation in postpar-
tum women. Indeed, after treadmill exercise, reduced
plasma cortisol responses were found in lactating compared
to non-lactating women (Altemus et al., 1995). Further-
more, suckling decreases basal levels of ACTH and total
plasma cortisol in lactating women (Chiodera et al., 1991;
Amico et al., 1994). Therefore, Heinrichs et al. (2001)
investigated whether a blunting of endocrine stress
responses in women can be ascribed to suckling as a
short-term effect or to lactation in general. Thus, lactating
mothers were randomly assigned either to breast-feed or to
hold their infants before they were exposed to the TSST.
While no significant differences in pre-stressor baseline
hormone levels could be observed between groups, salivary
and total plasma cortisol responses to stress were attenu-
ated in breast-feeding women. From these data, it can be
concluded that lactation in women, in contrast to rats, does
not result in a general restraint of HPA axis responses to
acute psychosocial stress. Rather, suckling seems to exert a
short-term suppression of the cortisol response to psycho-
social stress. In accordance with this finding others
described comparable total plasma cortisol responses to
psychosocial stress in lactating versus non-lactating women
(Altemus et al., 2001; Redwine et al., 2001). Finally, a
recent study raised the hypothesis that only multiple
repeats of the pregnancy/lactation period might modulate
HPA axis functioning since breast-feeding was associated
with reduced salivary cortisol stress responsiveness among
multiparous but not primiparous mothers (Tu et al., 2006). A
comprehensive review on lactation and stress and the
protective effects of breast-feeding in humans can be found
in Heinrichs et al. (2002), providing a detailed discussion of
the potential inhibitory impact of the lactogenic peptides
oxytocin and prolactin on different levels of HPA axis
2.4. Nicotine, coffee, alcohol and dietary energy
Nicotine is a potent acute stimulator of the HPA axis through
et al., 1989; Weidenfeld et al., 1989; Matta et al., 1998;
Rosecrans and Karin, 1998). After smoking at least two cigar-
ettes, smokers show significant elevations of salivary cortisol
levels (Kirschbaum et al., 1992d, 1994, 1997). Regular con-
sumption of nicotine could therefore lead to chronically
ness of the HPA axis to acute challenge. In fact, habitual
smoking changes the HPA axis responses to stress; blunted
salivary cortisol responses to the TSST have been repeatedly
observed in habitual smokers compared to non-smokers
(Kirschbaum et al., 1993b, 1994; al’Absi et al., 2003) while
nicotine abstinence did not alter salivary cortisol responses to
psychosocial stress in smokers (al’Absi et al., 2002, 2003).
responses between smokers and non-smokers have been
reported only after stimulation at a supra-pituitary level.
Injections of hCRH or bicycle ergometry resulted in no or only
marginally blunted salivary cortisol responses in chronic smo-
kers versus non-smokers (Kirschbaum et al., 1993b, 1994). In
the Dex-CRH-test, only ACTH responses showed positive
associations with nicotine consumption (Ku ¨nzel et al.,
2003). In sum, acute as well as habitual smoking is a potential
intervening variable which potentially accounts for some of
the inter- and intraindividual variation observed in salivary
cortisol responses to challenge (for review see Rohleder and
Beside nicotine, it has been shownthat caffeine intake can
potentially activate important components of the pituitary-
adrenocortical response in humans during resting states lead-
ing to increased plasma as well as salivary cortisol levels
(Lovallo et al., 1996, 2005). However, this finding is not
unequivocal since others did not observe increases in neither
basalfree nortotalcortisol levelsafter consumption ofcoffee
or tea (Quinlan et al., 1997; Lane et al., 2002; Lovallo et al.,
2006; Tsubouchi et al., 2006; MacKenzie et al., 2007; Steptoe
et al., 2007). In stress experiments evidence has emerged for
timetaskoranacademicexam onplasma and salivary cortisol
responses (Lane et al., 1990; al’Absi et al., 1995, 1998;
Shepard et al., 2000; Lovallo et al., 2006). Finally, caffeine
consumption was not related to HPA axis responses applying a
Dex-CRH-test, though salivary cortisol levels have not been
reported here (Ku ¨nzel et al., 2003).
There are several reports suggesting that chronic alcohol
consumption impacts on basal HPA axis activity as well as HPA
axis reactivity to stress (Gianoulakis et al., 2003; Adinoff
et al., 2005). Lovallo et al. (2000) observed blunted salivary
cortisol responses to a public speaking challenge in alcohol-
dependent patients compared to healthy controls, but others
could not find HPA axis response differences to the TSST
comparing alcoholicsor abstinent
subjects with non-alcoholics (Munro et al., 2005; McRae
et al., 2006). Consistently, in pharmacological stimulation
tests, reduced HPA axis responses to stimulation with hCRH
and Synacthen were repeatedly observed in chronic alco-
holics compared to non-alcoholics (Heuser et al., 1988; Costa
et al., 1996; Kearney et al., 2000; for review see Adinoff
et al., 2005). Also subjects with a positive family history of
plasma and salivary cortisol responses and an alcoholic drink
blunted subsequent stress-induced increases in plasma ACTH
and cortisol (Dai et al., 2002, 2007; Sorocco et al., 2006).
However, findings are again not unanimous. Zimmermann
et al. (2004) reported on elevated HPA axis stress response
Determinants of salivary cortisol responses to challenge7
to the TSST and a stronger dampening by alcohol in sons of
alcoholic fathers compared to control subjects. Such a heigh-
tened plasma cortisol response to a psychosocial stressor was
also described by Uhart et al. (2006) in Caucasian but not
African-American men and women with a positive family
history of alcoholism. In sum, chronic alcohol consumption,
a positive familiy history of alcohol dependence as well as
acute ethanol intake should be considered as potential fac-
tors impacting on salivary cortisol responses to acute stress.
Finally, the availability of dietary energy supplies appears
to exert important regulatory functions in pituitary-adrenal
stress responses pointing to an important role of the nutri-
tional state. In a first study, the effects of short-term fasting
and subsequent glucose administration on the salivary
cortisol response to the TSSTwere investigated (Kirschbaum
et al., 1997). Although glucose load per se did not affect
saliva cortisol levels, the stress exposure induced a large
salivary cortisol response in glucose-treated subjects. In
contrast, fasted subjects who received tap water did not
respond to the psychological stressor with significant changes
in salivary cortisol levels. The finding that low glucose levels
appear to inhibit adrenocortical responsiveness in healthy
subjects was rather surprising. In a second study, subjects
either received glucose, protein, fat, or water 1 h before
TSSTexposure (Gonzalez-Bono et al., 2002). Absolute saliva
cortisol levels and net increases were greater in the glucose
group in comparison to the other three groups and the
salivary cortisol response was positively correlated with
changes in blood glucose. In sum, it can be assumed that a
central mechanism may be responsible for regulation of
energy balance and HPA axis activation rather than periph-
eral mechanisms. These studies show that blood glucose
levels should be standardized when studying salivary cortisol
responses. This could be achieved by standardization of the
nutritional state before onset of an experiment, e.g. by the
consumption of a standardized meal or the administration of
a glucose-containing standard beverage at the beginning of a
stress experiment. Recently,Rohleder andKirschbaum(2007)
reviewed the effects of neuropeptides involved in energy
homeostasis and appetite regulation and concluded that
current evidence insufficiently explains the observed nega-
tive HPA axis modulation by low glucose levels since orexi-
genic peptidesstimulate the HPA axis rather than suppressing
it. Lastly, researchers should also be aware of potential
alterations of salivary cortisol responses to acute laboratory
stress due to intake of vitamin compounds (Brody et al.,
2.5. Genetic factors
Salivary cortisol responses to acute challenge are signifi-
cantly influenced by genetic factors as shown in twin studies
as well as in candidate gene studies on polymorphisms in HPA
mineralocorticoid or melanocortin 2 (ACTH) receptors (for a
review see Wu ¨st et al., 2004a). In a first pilot study, the
heritability of salivary cortisol responses was investigated in
a rather small and heterogeneous sample of monozygotic
(MZ) and dizygotic (DZ) twins who were exposed to the TSST,
an injection of 100 mg hCRH and exhausting physical exercise
(Kirschbaum et al., 1992b). While the results suggested a
substantial heritability of salivary cortisol peak levels after
hCRH administration, salivary cortisol responses to the TSST
an impact of genetic factors was detectable for responses to
bicycle ergometry. In a larger sample of young healthy male
twin pairs (33 MZ and 25 DZ) the TSSTwas performed three
times in 1-week intervals (Federenko et al., 2004). In accor-
dance with the hypothesis that ‘‘trait’’-like components of
the endocrine stress response may become more apparent
influence on HPA axis reactivity was low at the first TSST
stress exposure, but increased substantially with the repeti-
tion of the same stress protocol. This strongly supports the
notion that genetic factors do contribute to the variability in
salivary cortisol responses to psychosocial stress. While also a
significant heritability of salivary cortisol increases to 250 mg
synthetic ACTH (Synacthen) was found in this sample, sur-
prisingly no clear indication for a genetic effect on responses
to 1 mg Synacthen was found (Federenko, 2003).
More recently, in a series of studies Wu ¨st and coworkers
investigated for the first time whether variants of the
glucocorticoid or mineralocorticoid receptor gene might
contribute to the large interindividual variability of HPA axis
stress reactivity and amongst others they documented a
polymorphisms and salivary cortisol responses to acute
psychosocial stress (Wu ¨st et al., 2004b; DeRijk et al.,
2006; Kumsta et al., 2007b).
2.6. Methodological factors: habituation and
time of testing
From a methodological point of view, researchers should be
awareofthe factthata rapid habituationof a salivarycortisol
response after repeated exposure to (initially) stressful situa-
et al., 1989; Deinzer et al., 1997; Pruessner et al., 1997;
Schommer et al., 2003; Federenko et al., 2004; Kudielka
et al., 2006b). It has been hypothesized that such habituation
may be ascribed to a reduction in context variables across
stress sessions. Wu ¨st et al. (2005b) could recently document a
substantial interindividual variability of salivary cortisol
response habituation patterns. While 52% of the participants
showed the well-known response habituation to the TSST,
almost 16% showed a response sensitization across three test
sessions. Interestingly, such habituation effects appear to be
rather specific for HPA axis responses since parameters of
the sympathetic nervous system, the immune system, blood
coagulation system and indices of hemoconcentration showed
rather uniform activation patterns with repeated exposure to
psychosocial challenge (Schommer et al., 2003; von Ka ¨nel
et al., 2004, 2006; Mischler et al., 2005).
Another methodological issue relates to the time of
testing. Firstly, in case of early morning sessions, an experi-
menter should ensure that the onset of a stress experiment
does not interfere with the cortisol awakening response
(CAR). Secondly, we observed that absolute salivary cortisol
response curves after acute psychosocial stress exposure are
much higher in the morning although comparable net free
cortisol increases can be assessed with equal reliability in the
Numerous studies investigated the effect of time of day on
8 B.M. Kudielka et al.
HPA axis responses applying different provocation tests. The
majority of these studies conclude that maximum plasma
cortisol increases can be achieved in the afternoon; however,
salivary cortisol responses have not been measured in such
studies (for an overview see Kudielka et al., 2004b). After
physical exercise salivary cortisol responses appear not to
show a significant effect depending on time of day (Thuma
a greater rise in total cortisol in the evening than in the
morning, comparable increases emerged forsalivary cortisol.
2.7. Early life experiences: pre- and postnatal
There is first evidence that stressful pre- and postnatal life
experiences potentially exert a lifelong impact on HPA axis
responses to diverse psychological and pharmacological
challenge paradigms (Huizink et al., 2004; Luecken and
Lemery, 2004; Weinstock, 2005; Luecken and Appelhans,
2006; Entringer et al., in press). Fetal programming of the
HPA axis is proposed as one key mechanism underlying the
link between prenatal stress, adverse birth outcomes (par-
several diseases later in adult life. Indeed, birth weight was
inversely related to salivary cortisol responses to acute
psychosocial stress in male adults and boys (Wu ¨st et al.,
2005a; Jones et al., 2006). Salivary and plasma cortisol
responses to pharmacological stimulation have also been
shown to be significantly associated with birth weight and
gestational age (Kajantie et al., 2003; Ward et al., 2004).
in early childhood on salivary cortisol stress responses to
unfamiliar situations suggests a significant gene-environ-
ment interaction (Ouellet-Morin et al., 2008). Finally,
recent studies reported minor but significant relations
between childhood attachment styles and salivary cortisol
responses to acute stress during adulthood (Luecken, 1998,
2000; Quirin et al., 2008) or between adult couple attach-
ment styles and salivary cortisol responses in relationship
conflict situations (Powers et al., 2006). Other studies point
to significant interactional effects of psychiatric symptoms
in adult life and childhood trauma on HPA axis responses to
assessed during adulthood (Heim et al., 2000, 2008), though
salivary cortisol responses have not been reported in these
2.8. Social support, social hierarchy and
It is known that the social environment can exert modulating
effects on salivary cortisol stress responses. In men, brief
social support resulted in significantly decreased salivary
cortisol responses depending on the quality of support
whereas women showed even marginally higher salivary
cortisol responses when supported by their own partner in
life (Kirschbaum et al., 1995a). Heinrichs et al. (2003)
observed that the neuropeptide oxytocin enhances the buf-
fering effect of social support on salivary cortisol stress
responsiveness in men pointing to a potential underlying
biological mechanism for stress-protective effects of positive
social support. Also the position in the social hierarchy seems
to be relevant for acute salivary cortisol responses to acute
stress. In a sample of 63 army recruits, socially dominant
subjects showed high salivary cortisol increases compared
to only modest elevations in subordinate men after TSST
exposure and physical exercise (Hellhammer et al., 1997).
Besides the social environment, psychological interventions
like brief group-based cognitive-behavioral stress manage-
ment or relaxing music potentially reduce salivary cortisol
stress responses to an acute stress exposure and such effects
may persist over time in both men and women (Gaab et al.,
2003; Khalfa et al., 2003; Hammerfald et al., 2006).
2.9. Personality factors
It is tempting to speculate that salivary cortisol responses to
stress are influenced, at least in part, by stable trait factors
since the endocrine response to psychosocial stress can be
viewed as a close interaction between situation and person
variables within a given context. However, in most studies no
close relationship between personality factors and stress-
induced saliva cortisol increases could be observed. Though,
associations between personality traits and salivary cortisol
stress responses emerge after repeated stress exposures
(Kirschbaum et al., 1992a, 1995c; Pruessner et al., 1997).
While novelty may mask the impact of personality on the
salivary cortisol stress response on the first exposure, differ-
ences in the ability to cope with the stressful situation may
lead to different cortisol stress response patterns on subse-
quent stress exposures. Thus repeated stress exposures and
data aggregation seems to enhance the likelihood to find
stable and meaningful associations between personality vari-
ables and salivary cortisol stress responses.
2.10. Acute subjective-psychological stress
In humans, acute stress triggers multiple psychological as
well as physiological responses. Since such different
responses to a stressful event theoretically represent indi-
cators of the same construct, a strong association between
acute psychological and physiological responses, i.e. a high
psychoendocrine covariance, should be expected. However,
so far analyses of psychoendocrine covariance have produced
inconsistent and largely inconclusive results (for review see
Schlotz et al., 2008). Such inconsistencies might reflect
imperfect coupling of the different stress response systems.
Applying an innovative approach, Schlotz et al. (2008)
showed that earlier inconsistent findings are probably, at
least in part, based on the different dynamics of these
systems.While acute subjective
responses occur within seconds and may change dynamically
during a prolonged stress situation, cortisol responses reach
their peak approximately 15—20 min after the onset of the
esis that associations between an acute psychological and
endocrine stress response should be higher when response
correlations are computed at similar system-specific stages
relative to the onset of the stressor, the authors found that
responses and that high levels of cortisol are associated
with lower later levels of anxiety and activation using a
cross-correlational analytic approach. In sum, Schlotz and
Determinants of salivary cortisol responses to challenge9
coworkers demonstrated for the first time that time-lagged
conclusions about psychoendocrine covariance in response to
different stressors in humans.
2.11. Chronic stress, burnout and vital
The question whether an HPA axis hyper- or hyporesponsivity
to acute stress may occur when an individual is chronically
stressed or exhausted and no longer able to cope with
environmental stress is still open to debate. The few studies
that applied psychological stressors or even more seldom
pharmacological stimulation procedures potentially point at
a subtle free cortisol hyporeactivity in participants with
higher levels of chronic stress, burnout and exhaustion.
However, results are extremely heterogeneous with reports
on blunted as well as heightened free cortisol responses or no
differences in the HPA axis response to a single stress
exposure (for reviews see Kudielka et al., 2006a; Melamed
et al., 2006). It can only be speculated why the picture of
results is relatively heterogeneous. Inconsistencies between
studies can probably be, at least in part, ascribed to meth-
odological aspects (e.g., the use of different psychometric
scales and diagnostic criteria) as well as large differences in
the control of confounding or intervening factors of HPA axis
regulation. For instance, only some studies controlled for
psychological factors that might have influenced self-report,
like negative affectivity, depression or anxiety. It was also
hypothesizedthatchronic stress mayfirst lead tohyperactive
HPA axis functioning, while the system turns to hypoactive
functioning when a state of exhaustion is reached and the
individual is no longer able to cope with environmental stress
(for review see Kudielka et al., 2006a). Interestingly, under
repeated psychosocial short-term stress, an association
between vital exhaustion and salivary cortisol responses
emerged (Kudielka et al., 2006b). Linear regression revealed
a negative dose—response relationship between exhaustion
and the degree of response habituation to three TSST
exposures. With this, it can be assumed that situational or
psychological factors initially mask an existing impact
of exhaustion since an effect of exhaustion became only
apparent after repeated stress exposures.
2.12. Clinical studies and medication intake
It is well-known that HPA axis responses (including salivary
cortisol increases) to acute psychosocial stress as well as
different pharmacological stimulation tests are significantly
diseases to somatic complaints. To date, the TSST has been
applied in patients suffering from major depression, anxiety
disorder, social phobia, posttraumatic stress disorder, atten-
tion deficit hyperactivity disorder, chronic fatigue syndrome,
fibromyalgia, diverse pain disorders, functional gastrointest-
inal disorders, diabetes, different manifestations of chronic
cancer survivors, to give some examples. A detailed descrip-
and numerous reviews on this topic can be found elsewhere
(Holsboer, 1989; Heuser et al., 1994a; Tsigos and Chrousos,
and Hellhammer, 2003; Burke et al., 2005; Kudielka et al.,
2007c; Jessop and Turner-Cobb, 2008). Irrespective of
the heterogeneity of findings and evident methodological
differences across studies, results show that differences
between patients and healthy controls are more likely
observed when the system is challenged.
Finally, researchers should be aware of the fact that even
short-term medication (e.g., with glucocorticoids, psychoac-
tive drugs, or other pharmaceuticals ingested for preventive
reasons) potentially alters salivary cortisol responses to
psychosocial stress or pharmacological stimulation in patients
as well as healthy controls although free cortisol baseline
levels might remain unchanged (Tunn et al., 1992; Makatsori
et al., 2004; Pariante et al., 2004; Maheu et al., 2005; Fries
etal., 2006;Kudielka etal.,2007a).Likewise,treatmentwith
responses to Dex-CRH as shownin several studies, though free
cortisol responses have not been measured in such studies
(Ku ¨nzel et al., 2003; Rinne et al., 2003; Schu ¨le et al., 2003;
lying mechanisms how such medication impacts on HPA axis
stress regulationcanbefoundelsewhere(Bardenetal., 1995;
Holsboer and Barden, 1996; Holsboer, 2000; Pariante and
3. Final remarks
Therefore, understanding determinants of inter- and intrain-
underlying pathologically relevant dysregulation of cortisol
activity is a key topic in psychobiological stress research.
Evidence from research over the last decades clearly docu-
ments that salivary cortisol is a useful and valid biomarker in
stress research (see Hellhammer et al., in press). Amongst
and intervening factors, carefully described in different
laboratories and summarized in the present paper, can have
an impact on cortisol responses. This is certainly a somewhat
challenging situation as it is not possible to control for
countless potential confounders in each study and this holds
is the rule rather than the exception in our field. However,
it first should be noted that most of these factors do not
specifically modulate salivary cortisol but also cortisol
levels in blood. In general, the experience that the visible
complexity of a system increases with
into the system is a rather familiar experience for many
research disciplines. Moreover, the awareness of modulators
certainly offers the opportunity to reliably detect effects
and to improve, technically spoken, the signal to noise
ratio. This is crucial as the effects that can be expected in
psychobiological stress research are usually of modest size
(although they can well be of psychobiological or clinical
Thus, our knowledge might be helpful at different stages
of a research project. First, when planning an experiment, it
might guide the researchers’ decision on exclusion criteria,
eligibility and selection of subjects (depending on the study
question), further information that should preferably be
provided by participants (e.g., assessed in accompanying
demographic or psychometric assessments), factors that
10B.M. Kudielka et al.
could be held constant across subjects, and issues that are
relevant for the instruction of subjects before and during
the assessment period (e.g., subjects’ coffee consumption
at test days). For example, smoking or the intake of oral
contraceptives can be defined as exclusion criteria, can be
held constant across different study groups, can afterwards
be used as covariate, or defined as the experimental manipu-
lation in a (quasi-) experimental study design. Such decisions
the sample size should be based on a priori calculations (e.g.,
with the software ‘‘GPower3’’ by Faul et al., 2007, which is
freely accessible at http://www.psycho.uni-duesseldorf.de/
abteilungen/aap/gpower3). Due to the large variability of
possible research questions and study designs, it is difficult to
give specific recommendations. However, we think that at
least two factors should be considered in the vast majority of
studies which include the assessment of cortisol responses to
challenge. The first one is the time of day when the subjects
are exposed to the challenge and in most studies it should be
doable to hold this variable relatively constant. The most
convenient time window is the late afternoon. The second
prime variable is the subjects’ sex. It is often tempting to
restrict the sample to males (as it is done in most rodent
studies in our area) and this is surely an acceptable approach
to control for this factor. However, given the increasing
awareness of the striking quantity of sex-related influences
on brain function and disease vulnerability (for a recent
review see Cahill, 2006) males and females should be
included whenever possible. The gold standard to account
for the potential impact of the menstrual cycle on salivary
cortisol responses in women is to assess the subject’s cycle
phase and to study women in the follicular and women in the
luteal phase (repeated measures or group design depending
on the study). Focusing on only one menstrual cycle phase or
studying only women using oral contraceptives (OC) are less
laborious alternatives but both approaches result in a limited
generalizability of findings. It could be argued that particu-
larly OC intake is a clear artificial state and should thus be a
general exclusion criterion. However, in many countries the
majority of (young) women use OCs. Thus, investigating
female cortisol responses in front of the background of
widespread OC usage is — to a certain degree — a research
question initsownright.In thisrespect, itisalsoobviousthat
we need to consider a female’s pubertal or menopause
Second, when it comes to data analysis, knowledge about
moderating and intervening factors can help to select (or
test) a set of potentially (or the most) relevant control
variables to be used as covariates in statistical models.
However, researchers should be aware of the fact that the
appropriatenumber ofcovariates dependsonthesample size
since model overfitting might lead to spurious results
(Babyak, 2004). In general, the larger the available sample
size, the more variables can be added to a respective model.
Third, the acknowledgement of potential sources of vari-
ance is finally essential when it comes to data interpretation.
This might be especially important, for example, for studies
based on small sample sizes, quasi-experimental designs,
studies with limitations in randomization, or studies con-
ducted under ambulatory settings and field conditions, etc. A
discussion of potential sources of variance might contribute
to the explanation of contradictory or conflicting results
across different studies and might trigger the exploration
of further yet unknown sources of variance. Another impor-
tant aspect is the issue of generalizability of given results.
For example, if a study examines exclusively middle-aged
the results might not apply to females or other age cohorts,
resulting in reduced generalizability (see above). Finally, in
case of secondary analyses and reanalyses that take advan-
tage of preexisting samples or data sets, researchers should
be particularly aware of potential sources of inter- as well as
intraindividual variance since a given sample might have very
special characteristics due to the original study aim partici-
pants were recruited for. However, we should also bear in
mind that even in highly controlled studies results might be
sample-specific forunknown reasons.Therefore, replications
in diverse study samples are always a necessary requirement.
Meanwhile, clinical studies continue to accumulate
evidence that different diseases are associated with
characteristic salivary cortisol stress response profiles.
Furthermore, biological mechanisms begin to unfold which
could be helpful in explaining stress-disease associations.
Finally, the question how HPA axis challenge paradigms
that are capable to elicit acute salivary cortisol responses
(including psychosocial stress protocols like the Trier Social
Stress Test) can be applied as a diagnostic tool for the
prediction of disease susceptibility and symptom severity
and/or for monitoring the efficacy of interventions still
remains an important area of research for the future.
Role of the funding sources
1401/4-1, KU 1401/4-2, and KU 1401/4-3 of the German
Research Foundation (DFG) awarded to Brigitte M. Kudielka.
BMK, SWand DHH are members of the International Research
Training Group IRTG funded by the DFG (GRH 1389/1). The
DFG had no further role in writing the report and decision to
submit the paper for publication.
Conflict of interest
This work was carried out while all authors were affiliated
with the Graduate School of Psychobiology, Department of
Theoretical and Clinical Psychobiology, University of Trier,
Johanniterufer 15, 54290 Trier, Germany.
This work was supported by Emmy Noether research grant KU
1401/4-1, KU 1401/4-2, and KU 1401/4-3 of the German
Research Foundation (DFG) awarded to Brigitte M. Kudielka
as well as the International Research Training Group IRTG
funded by the DFG (GRH 1389/1; BMK, DHH, and SW are
members of the IRTG).
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