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RESEARCH ARTICLE
Stress Effects on Mood, HPA Axis, and
Autonomic Response: Comparison of Three
Psychosocial Stress Paradigms
Grace E. Giles*, Caroline R. Mahoney, Tad T. Brunye´, Holly A. Taylor,
Robin B. Kanarek
Department of Psychology, Tufts University, Medford, Massachusetts, United States of America
*grace.giles@tufts.edu
Abstract
Extensive experimental psychology research has attempted to parse the complex
relationship between psychosocial stress, mood, cognitive performance, and
physiological changes. To do so, it is necessary to have effective, validated
methods to experimentally induce psychosocial stress. The Trier Social Stress Test
(TSST) is the most commonly used method of experimentally inducing
psychosocial stress, but it is resource intensive. Less resource intense
psychosocial stress tasks include the Socially Evaluative Cold Pressor Task
(SECPT) and a computerized mental arithmetic task (MAT). These tasks effectively
produce a physiological and psychological stress response and have the benefits of
requiring fewer experimenters and affording data collection from multiple
participants simultaneously. The objective of this study was to compare the
magnitude and duration of these three experimental psychosocial stress induction
paradigms. On each of four separate days, participants completed either a control
non-stressful task or one of the three experimental stressors: the TSST, SECPT, or
MAT. We measured mood, working memory performance, salivary cortisol and
alpha-amylase (AA), and heart rate. The TSST and SECPT exerted the most robust
effects on mood and physiological measures. TSST effects were generally evident
immediately post-stress as well as 10- and 20-minutes after stress cessation,
whereas SECPT effects were generally limited to the duration of the stressor. The
stress duration is a key determinant when planning a study that utilizes an
experimental stressor, as researchers may be interested in collecting dependent
measures prior to stress cessation. In this way, the TSST would allow the
investigator a longer window to administer tasks of interest.
OPEN ACCESS
Citation: Giles GE, Mahoney CR, Brunye´TT,
Taylor HA, Kanarek RB (2014) Stress Effects on
Mood, HPA Axis, and Autonomic Response:
Comparison of Three Psychosocial Stress
Paradigms. PLoS ONE 9(12): e113618. doi:10.
1371/journal.pone.0113618
Editor: Alexandra Kavushansky, Technion - Israel
Institute of Technology, Israel
Received: May 29, 2014
Accepted: October 27, 2014
Published: December 12, 2014
Copyright: ß2014 Giles et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and repro-
duction in any medium, provided the original author
and source are credited.
Data Availability: The authors confirm that all data
underlying the findings are fully available without
restriction. All relevant data files are available from
the Dryad Repository: http://doi.org/10.5061/dryad.
64313.
Funding: The authors have no support or funding
to report.
Competing Interests: The authors have declared
that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0113618 December 12, 2014 1/19
Introduction
Psychological stress influences numerous psychological and physical processes in
both healthy individuals and those with psychiatric disorders [1–3]. Stress is
thought to influence mood [4,5], memory [6], and decision-making [7]. The
effects of psychological stress are physical as well, in that acute stress activates the
hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system
(SNS), producing elevations in cortisol, alpha-amylase (AA), and heart rate [8,9].
Extensive experimental psychology research has attempted to parse the complex
relationship between psychosocial stress, mood, cognitive performance, and
physiological changes. For instance, understanding how acute stressors influence
SNS response and performance on perceptual and cognitive tasks. Unfortunately
much of this research proceeds without empirical understandings of how acute
stress inductions may vary in their efficacy and duration. The present research was
aimed at providing a first baseline understanding of how three commonly used
stress inductions influence physiological, affective, and cognitive responding.
Efficacy of experimental stress paradigms
The Trier Social Stress Test (TSST) [10] is the most commonly used method of
experimentally inducing psychosocial stress. The TSST consists of three-parts, a
10-minute preparatory stage, 5-minute speech, and 5-minute mental arithmetic
task, all in front of a panel of investigators, purportedly trained in analyzing
nonverbal behavior [10]. Its socioevaluative and anticipatory components are
thought to contribute to its efficacy as a stressor. The TSST is thought to be an
effective method for inducing psychosocial stress, as it has repeatedly been found
to increase anxiety, cortisol, and AA [11–13]. It also influences performance on a
number of cognitive domains, including declarative memory (particularly for
emotionally-arousing material), spatial, and working memory [14–16], as well as
decision-making [7]. However, the TSST has a logistical limitation in that only
one participant can be tested at a time and for each participant, numerous
experimenters are required to staff the ‘‘panel.’’
More recent and less resource intensive psychosocial stress tasks include the
Socially Evaluative Cold Pressor Task (SECPT) [17–19] and computerized mental
arithmetic task (MAT) [20,21]. These tasks effectively produce a physiological
and psychological stress response and have the benefit of requiring fewer
experimenters and, in the case of the MAT, being able to run concurrent
participants. However, it is unclear how these three tasks compare in terms of the
magnitude and duration of the stress response.
Both cortisol and AA increase following onset of acute stressors, however
whereas cortisol levels peak at 10–30 minutes after cessation of stressor and take
approximately 90 minutes to return to baseline, AA levels peak 5–10 minutes
post-stressor and take only 10–15 minutes to return to baseline [1,22,23].
Although multiple studies have assessed the relative time course of cortisol and
AA levels before and up to one hour following acute stress [9,22,24], to our
Comparison of Three Psychosocial Stress Paradigms
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knowledge no study has assessed changes in mood and behavior beyond
immediate post-stress induction. Therefore, it is not known how, exactly, the time
course of changes in physiological indices of stress, such as cortisol and AA levels,
relate to changes in mood and behavior.
In designing a study to analyze the effects of stress on a given cognitive
function, it is necessary to know the duration of physiological and behavioral
effects associated with the stressor (i.e. if stress-induced changes of the TSST are
only significantly different from baseline for 30-minutes post-stress induction, the
study should be designed such that any critical measures occur within that 30-
minute window). Despite the practical importance of this knowledge, no study to
date has assessed the differential magnitude and duration of experimental stress
paradigms in a controlled, within-subjects design.
Stress effects on working memory
Stress influences memory through the release of cortisol, which binds to
glucocorticoid (GC) receptors located in the hippocampus and prefrontal cortex
(PFC), making these two regions particularly vulnerable to stress [25]. Multiple
studies have documented the influence of stress on hippocampal-dependent
declarative memory, especially for emotionally arousing material [3]. However,
the literature to date on prefrontal-dependent working memory is less consistent.
Working memory refers to the limited capacity system in which information is
temporarily stored, updated, and maintained [26]. While some studies have found
that stress impairs working memory performance [15,17,18,27,28] others found
no effects [17,29].
We chose to examine stress-induced working memory changes using the N-
Back Task, which challenges working memory by having participants monitor a
series of briefly presented stimuli and decide on each trial if the current stimulus is
the same as the one presented one, two or three trials before. The task emphasizes
working memory monitoring and constant updating. The current design utilizes
spatial cues at three levels of task load (1-back, 2-back, and 3-back) [30].
The present study
The objective of this study was to compare the magnitude and duration of three
experimental psychosocial stress induction paradigms. On each of four separate
days, participants completed either a control non-stressful task or one of three
experimental stressors: the TSST, SECPT, or MAT. We measured physiological
response by collecting heart rate data throughout each test session. Participants
first completed baseline salivary cortisol, AA, and affective state measures,
followed by the stress induction. Following the stressor, participants completed
three consecutive test blocks lasting 10 minutes each, consisting of affective
questionnaires, a working memory task, and salivary cortisol and AA collection.
We hypothesized that all experimental stressors would increase negative affect
ratings, increase salivary cortisol and AA levels, and impair working memory
Comparison of Three Psychosocial Stress Paradigms
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performance. We also predicted that these effects would be most pronounced
within conditions in the first test block (immediately following the stressor) and
between conditions following the TSST and SECPT, which have the most
pronounced socioevaluative components [31].
Materials and Methods
Participants
Twenty four undergraduate students (7 male; mean age 20. 63¡1.91 years; mean
BMI 20.91¡2.89) participated for monetary compensation ($10 USD/hr). All
participants were non-nicotine users and did not use prescription medication
other than oral contraceptives. Exclusion criteria also included being pregnant or
nursing, having a history of depression, anxiety disorders, panic attacks, cardiac
disease, hypertension, or insomnia. Written informed consent was obtained, and
all procedures were approved by the Tufts University Institutional Review Board.
Design
We used a repeated measures design with four levels of our independent variable,
Stress (TSST, SECPT, MAT, Control Task). Condition order was fully counter-
balanced across participants.
Profile of Mood States Questionnaire
The Profile of Mood States (POMS) is an inventory of subjective mood and
arousal [32]. Participants rate a series of 65 mood related adjectives on a 5-point
scale, using the response set of ‘‘how are you feeling right now?’’ The adjectives
factor into six mood subscales (tension, depression, anger, vigor, fatigue, and
confusion). The POMS is sensitive to a wide range of environmental factors; sleep
loss, nutritional manipulations, and sub-clinical doses of various drugs [4,33,34].
N-Back Task
The N-Back Task challenges working memory by having participants monitor a
series of briefly presented stimuli and decide on each trial if the current stimulus is
the same as the one presented one, two or three trials before. This task emphasizes
working memory monitoring and constant updating. The current design utilizes a
spatial N-Back Task, which involves monitoring object locations in different
screen regions, each at three levels of task load: 1-back, 2-back, and 3-back [30].
Participants completed 57 trials within each load (total 171). Each stimulus was
presented for 500 ms followed by a blank screen (2500 ms). Participants could
respond either during the stimulus presentation or blank screen. Dependent
measures include response time, hit rate, and sensitivity (d9).
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Arousal Measures
Salivary Cortisol and Alpha-Amylase
Saliva was collected for salivary cortisol and alpha-amylase (AA) analyses
(biomarkers for arousal) using the SalivaBio Oral Swab (SOS) Method.
Participants placed a swab under their tongue for 2 minutes. Swabs were placed
into 1.8 ml plastic vials and immediately stored at -20˚C (or colder) until assayed.
Samples were analyzed in duplicate for cortisol and in singlet for AA in an
independent laboratory (Salimetrics LLC, State College, Pennsylvania).
Heart rate
Heart rate data was collected using Equivital heart rate monitors. The monitor
consisted of a transmitter worn against the skin and around the chest. The
transmitters picked up and stored temporarily signals from the participant’s heart
and skin. The data was downloaded at the end of each experimental session.
Participants were instructed on the proper placement of the heart rate strap and
then asked to don the strap and sensor themselves. The experimenter then
confirmed the signal.
Stress Conditions
Trier Social Stress Test (TSST)
The TSST is a 20-minute psychosocial stress task consisting of 3 stages: (1) 10-
minute preparatory stage, (2) 5-minute public speaking task, and (3) 5-minute
mental arithmetic task [10]. In the first stage, participants were led into a
conference room and introduced to a panel of three experimenters. They were
given 10 minutes to prepare a 5-minute mock job-talk that would be videotaped
and assessed for nonverbal behavior and voice frequency. In the second stage,
participants delivered the 5-minute speech. If they ended in less than 5 minutes,
they were asked to continue talking. In the third stage, participants completed a
mental arithmetic task, in which they serially subtracted a prime number from a
4-digit number (e.g. 17 from 1223) and had to start over if they made a mistake.
Socially Evaluative Cold Pressor Test (SECPT)
In the SECPT, participants were led into a conference room. An experimenter
explained that they would immerse their arm, up to the elbow, in ice water (4˚C)
for up to 3 minutes, and that they would be videotaped to later assess their facial
expression (Fig. 1). Participants were told that they could remove their hand at
any time, and were told when the 3 minutes had elapsed [19].
Arithmetic Task (MAT)
Participants performed a mental arithmetic for 20 minutes, similar to Kimura
et al., 2006. Participants completed simple arithmetic problems presented one at a
time on the computer monitor. Each problem appeared for 1000 ms and the
participant had 1500 ms to respond using a standard numeric keypad. Feedback
(correct/incorrect) was provided immediately following each response. Before
beginning, participants were told that while actual performance varies on the task,
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the average performance of their peers for the given set of arithmetic problems is
approximately 54% (as derived from pilot participants). Critically, to induce an
acute psychosocial stress, the system adjusted problem difficulty to maintain an
experimenter-set performance level below the ‘‘average student’’ performance
level.
Control Stress Task
Participants completed a 20-minute neutral arithmetic task, similar to the stressful
MAT, with the exception that the system adjusted problem difficulty to maintain
an experimenter-set performance level approximately equal to ‘‘average student’’
performance of the participants’ peers.
Procedure
Participants completed five sessions on separate days: one practice session to
become familiarized with the experimental procedure and tasks, and four test
sessions corresponding to each Stress condition. During the practice session,
participants completed screening materials and signed the informed consent. They
were then familiarized with test procedures, including putting on the heart rate
monitor, saliva collection and questionnaires. They then completed POMS. They
then received instructions for the N-Back Task and completed practice trials. In
addition, height and weight were taken. The practice and test sessions took place
in the afternoon; beginning between 1300–1500 h. To control for potential effects
of circadian rhythm, start time was consistent within each participant.
During test sessions, participants donned the heart rate strap and completed
baseline measures of the POMS and saliva sample. Participants then completed
the stress induction or non-stressful control task. After the stress induction they
again completed the questionnaires and the N-Back Task, followed by a second
saliva sample. The questionnaires, N-Back Task, and saliva sample constituted 1
Fig. 1. Socially Evaluative Cold Pressor Test (SECPT). Image represents the SECPT, in which participants
immerse their arm, up to the elbow, in ice water (4˚C) for up to 3 minutes, in front of a videocamera and
experimenter.
doi:10.1371/journal.pone.0113618.g001
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block of the test battery. The participants completed 3 blocks in succession (see
Fig. 2 for schematic of procedure).
Statistics
The POMS and salivary measures were analyzed using an Analyses of Variance
(ANOVA) with Stress condition (TSST, SECPT, MAT, Control Task) and Time
(baseline and 0, 10-, and 20-minutes post-stress) as within-participants factors.
Analysis of the N-Back Task was conducted using an ANOVA with Stress
condition (TSST, SECPT, MAT, Control Task), Load (1-, 2-, and 3-Back) and
Time (0-, 10-, and 20-minutes post-stress) as within-participants factors. Analyses
of the heart rate data were conducted in the same manner with Time divided into
the following 5 intervals: baseline, stress induction, 0-, 10-, and 20-minutes post-
stress).
Salivary AA concentrations were first adjusted for salivary flow rate, calculated
as Saliva weight gðÞ
Time minutesðÞ
|AA U
ml
where Time equals two minutes. Adjustment for
salivary flow rate is necessary given that the output of AA per unit time, not its
concentration, is associated with the sympathetic stress response [35]. Because
similar studies found that salivary cortisol and AA concentrations had skewed
distributions [15], we tested for normality using the Lilliefors procedure. Cortisol
and AA data showed a positively skewed distribution, and therefore were log-
transformed. The ANOVAs were performed with the transformed data; for
comprehensibility, only pre-transform data are presented in figures and tables.
After removing outlying values, cortisol data reflects n 519 subjects and AA data
reflects n523 subjects. In order to examine pre-stress differences, an additional
ANOVA compared the four Stress conditions at baseline, i.e. before subjects were
informed of the particular days’ condition.
An effect was deemed statistically significant if the likelihood of its occurrence
by chance was p,0.05. When sphericity was violated, Greenhouse–Geisser
corrected p-values were used. When an ANOVA yielded a significant main effect,
Fig. 2. Schematic representation of the study schedule. During the study sessions, participants first
completed baseline measures of the POMS and provided saliva samples for analysis of cortisol (CORT) and
alpha-amylase (AA). They completed one of three experimental stressors, or the non-stressful control task.
They then completed three blocks in succession, each consisting of the POMS, salivary measures, and a
spatial N-Back task.
doi:10.1371/journal.pone.0113618.g002
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post-hoc tests using the Bonferroni correction were conducted. All statistical
analyses were performed using SPSS 12.0.
Results
Stress Administration Order
The order of the Stress conditions was counterbalanced across participants to
circumvent order effects. Four Stress conditions resulted in 24 possible orders,
meaning each subjects was tested with a unique order. It was not feasible to fully
assess order effects, given that each order would contain n 51. Nonetheless, all
measures were subjected to analyses testing whether the First Condition
completed influenced results. With two minor exceptions, no main effects or
interactions were found with First Condition on the POMS, physiological
measures, or N-Back task (all ps.0.16). A significant Load x First Task
interaction on N-Back hit rate F(2,40) 52.417, p,0.05 showed that response
time was progressively higher as load increased when the SECPT, MAT and
Control Tasks were administered first (ps,0.01), but not when the TSST was
administered first (p.0.16). Further, a marginal Stress Condition x First Task
interaction on heart rate F(9,60) 51.768, p,0.1 was found but did not yield any
significant effects on follow up tests (all ps..11).
Profile of Mood States (POMS)
The six mood subscales of the POMS were analyzed separately: tension,
depression, anger, vigor, fatigue, and confusion, as well as total mood disturbance
[32]. No effects were found for anger or depression. Analysis of the tension
subscale revealed a main effect of Time F(3,69) 57.816, p,0.01 in which feelings
of tension were significantly higher than baseline immediately post-stress and
significantly lower than baseline at 10- and 20-minutes post-stress. A Stress x
Time interaction F(9,207) 55.074, p,0.01 revealed that feelings of tension were
significantly higher than baseline at all time points following the TSST F(3,69) 5
12.739, p,0.01, but were not significantly different following the other three tasks
(Table 1). Analyses comparing the stress tasks to the control tasks showed that
relative to the control task, feelings of tension were higher following the TSST
immediately post-stress only F(3,69)56.998, p,0.01, but no effects were found
for the SECPT, MAT or other time points.
Analysis of the vigor subscale revealed a main effect of Time F(3,69) 521.386,
p,0.01, in which vigor was significantly higher at baseline than all other time
points.
Analysis of the fatigue subscale revealed a main effect of Stress F(3,69) 53.869
p,0.05 in which feelings of fatigue were significantly lower during the TSST and
SECPT than the control condition. A main effect of Time F(3,69) 55.043,
p,0.01 indicated that fatigue was higher 20-minute post-stress than at baseline.
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Analysis of the confusion subscale revealed a main effect of Time F(3,69) 5
3.617, p,0.05, in which feelings of confusion were significantly higher 20-minutes
post-stress than at baseline.
Analysis of the total mood disturbance subscale revealed main effect of Time
F(3,69) 58.694, p,0.01 in which total mood disturbance was significantly higher
than baseline at all other time points, and a Stress x Time interaction F(9,207) 5
3.287, p,0.05, in which total mood disturbance was higher than baseline at all
three time points following the TSST F(3,69) 57.880 p,0.01 and MAT F(3,69) 5
7.151, p,0.01. No effects were found for the control condition or SECPT.
Analyses comparing the stress tasks to the control tasks revealed no differences.
Table 1. Profile of Mood States (POMS) as a function of stress (means and SE) at baseline and 0-, 10- and 20-minutes post-stress.
Adjective Time TSST SECPT Math Control
Tension Baseline 1.17 (1.07) 1.79 (1.05) 2.17 (1.28) 2.71 (1.18)
0-min 7.13** (1.60) 2.21 (0.88) 4.21 (1.11) 3.38 (1.05)
10-min 3.79** (1.37) 2.33 (1.11) 2.71 (1.08) 4.13 (1.35)
20-min 4.08** (1.35) 2.50 (1.04) 2.71 (1.03) 3.96 (1.17)
Depression Baseline 15.33 (.630) 3.04 (0.97) 12.96 (0.67) 5.33 (1.47)
0-min 11.96 (1.440) 2.96 (0.92) 10.71 (0.86) 3.58 (1.05)
10-min 10.21 (1.132) 5.17 (1.98) 9.46 (1.01) 5.42 (1.71)
20-min 10.17 (1.283) 4.92 (1.58) 9.21 (0.88) 4.75 (1.67)
Anger Baseline 3.33 (1.13) 2.25 (0.63) 4.75 (0.79) 3.50 (1.04)
0-min 3.58 (1.61) 2.79 (0.93) 6.13 (1.01) 3.67 (1.17)
10-min 4.38 (1.46) 3.92 (1.61) 5.67 (0.81) 4.88 (1.58)
20-min 5.17 (1.33) 3.38 (1.27) 5.88 (0.97) 5.08 (1.53)
Vigor Baseline 1.33 (1.32) 14.21 (0.92) 1.13 (1.48) 11.17 (1.18)
0-min 2.29 (1.52) 13.38 (1.04) 2.33 (1.52) 10.75 (1.29)
10-min 2.38 (1.49) 11.04 (1.31) 2.13 (1.44) 9.58 (1.38)
20-min 2.92 (1.40) 9.79 (1.18) 2.63 (1.43) 9.13 (1.46)
Fatigue Baseline 24.75 (1.12) 3.58 (0.93) 20.21 (1.27) 6.67 (1.21)
0-min 10.25** (1.01) 3.38 (0.82) 9.63* (1.42) 5.17 (0.96)
10-min 8.71** (1.23) 4.29 (1.17) 7.83* (1.41) 7.00 (1.28)
20-min 11.04** (1.33) 5.75 (1.32) 9.00* (1.29) 7.21 (1.29)
Confusion Baseline 1.17 (0.98) 1.00 (0.79) 2.17 (0.85) 2.63 (0.91)
0-min 7.13** (0.95) 1.42 (0.90) 4.21 (1.02) 1.63 (0.84)
10-min 3.79** (1.03) 2.54 (1.21) 2.71 (0.90) 2.17 (0.97)
20-min 4.08** (1.02) 3.00 (1.18) 2.71 (0.98) 2.58 (0.85)
Total Mood Baseline 15.33 (4.68) 22.54 (3.89) 12.96 (5.03) 9.67 (5.65)
0-min 11.96 (6.41) 20.62 (4.27) 10.71 (5.52) 6.67 (4.88)
10-min 10.21 (5.94) 7.21 (6.86) 9.46 (5.36) 14.21 (6.42)
20-min 10.17 (6.13) 9.75 (5.81) 9.21 (5.54) 14.25 (6.08)
*p,0.05, **p,0.01, levels of significance relative to baseline in stress x time interactions. Additionally, we found a main effect of the type of stress on the
fatigue subscale, in which feelings of fatigue were lower during the TSST and SECPT than control condition p,0.05.
doi:10.1371/journal.pone.0113618.t001
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N-Back Task (NB)
Dependent measures on the N-Back Task included response time (Table 2), hit
rate (Table 3), and sensitivity (d9;Table 4). Sensitivity is a composite index of hit
rate and false alarm rate, which was calculated by subtracting the z-score of the
false alarm rate from the z-score of the hit rate. Response time data reflect n 523,
as one subject responded with a Hit Rate of 0 during 1 block of trials. Across all
conditions, analyses revealed main effects of Load (all p,0.01), in which hit rate
and d9were greater in the 1-back and 2-back than 3-back loads, and response time
was lower in the 1-back and 2-back compared to 3-back load. A main effect on
Time on response time showed that response time decreased across the three
iterations of the task (ps,0.05). A marginal Stress x Time interaction F(6,138) 5
1.884 (p,0.1) indicated that response times were higher immediately following
the TSST (p,0.05) than 10- and 20 minutes after, and marginally lower
immediately after the Control (p,0.1) than 10- and 20 minutes after, but no
differences were found following the SECPT or MAT. No further effects were
found for Stress, Time, or Stress x Time interactions (all ps.0.28).
Physiological Measures
Heart Rate
Results revealed a main effect of Time, F(4,92) 543.729, p,0.01, in which heart
rate was significantly higher than baseline during the stress induction, and lower
than baseline at all time-points post-stress (Fig. 3). A Stress x Time interaction
F(12,276) 54.624, p,0.01 showed a main effect of Time in the TSST, SECPT,
and Control conditions, but not the MAT. In the TSST, heart rate was higher than
baseline during the stress induction and lower than baseline 10-30 min post-stress
F(4,92) 529.526, p,0.01 (no effects found 0-10 min post-stress). In the SECPT,
heart rate was higher than baseline during the stress induction and lower than
baseline at all time-points post-stress F(4,92) 531.379, p,0.01. During the
Table 2. N-Back Reaction Time (seconds) as a function of Stress and Load (means and SE) at baseline and 0-, 10- and 20-minutes post-stress.
Time Load TSST SECPT MAT Control
0 Min 1 723.63 (40.31) 722.18 (25.63) 701.57 (27.47) 705.97 (29.30)
2 780.98 (50.28) 771.82 (32.34) 748.97 (29.34) 772.73 (47.10)
3 793.38 (50.32) 763.38 (41.23) 737.55 (39.86) 760.52 (57.48)
10 Min 1 637.90 (25.24) 685.75 (20.99) 688.84 (28.68) 701.42 (29.54)
2 721.31 (39.39) 774.79 (37.95) 716.03 (32.32) 779.72 (50.51)
3 747.11 (39.65) 760.93 (52.05) 773.67 (34.57) 762.58 (43.66)
20 Min 1 667.04 (35.02) 670.79 (34.86) 681.82 (25.18) 701.25 (31.89)
2 736.56 (44.25) 695.29 (30.24) 716.14 (42.37) 780.35 (44.84)
3 738.01 (38.35) 716.71 (41.96) 717.72 (41.65) 779.68 (48.07)
No significant effects were found for Stress, Time, Load or any interactions.
doi:10.1371/journal.pone.0113618.t002
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control condition, heart rate was lower than baseline during all time points post-
stress F(4,92) 59.121, p,0.01.
Salivary Cortisol
No baseline differences between the four Stress conditions were found (all
ps..35). Analyses of salivary cortisol revealed a Stress x Time interaction F(9,162)
53.894, p,0.01, in which cortisol levels were higher than baseline at all time-
points post-stressor following the TSST F(3,54) 54.921, p,0.01 (Fig. 4). No
differences were found for the SECPT, MAT or control task (all ps..08). Analyses
comparing the stress tasks to the control tasks at each time point showed that
cortisol levels were higher in the TSST and SECPT conditions 20-minutes post-
stress F(3,57)56.203, p,0.05. No differences were found for the MAT or for any
stressors pre-stress, or immediately or 10 minutes post-stress (all ps..06).
Table 3. N-Back Hit Rate as a function of Stress and Load (means and SE) at baseline and 0-, 10- and 20-minutes post-stress.
Time Load TSST SECPT MAT Control
0 Min 1 0.77 (0.04) 0.78 (0.04) 0.76 (0.05) 0.72 (0.05)
2 0.68 (0.04) 0.69 (0.04) 0.64 (0.04) 0.64 (0.04)
3 0.49 (0.03) 0.47 (0.04) 0.53 (0.04) 0.48 (0.03)
10 Min 1 0.73 (0.05) 0.76 (0.04) 0.76 (0.04) 0.71 (0.05)
2 0.69 (0.04) 0.66 (0.04) 0.67 (0.04) 0.67 (0.04)
3 0.49 (0.04) 0.48 (0.04) 0.50 (0.04) 0.48 (0.03)
20 Min 1 0.74 (0.04) 0.73 (0.05) 0.75 (0.05) 0.68 (0.06)
2 0.65 (0.05) 0.66 (0.05) 0.66 (0.04) 0.68 (0.04)
3 0.51 (0.04) 0.49 (0.04) 0.45 (0.04) 0.51 (0.03)
No significant effects were found for Stress, Time, Load or any interactions.
doi:10.1371/journal.pone.0113618.t003
Table 4. N-Back Sensitivity (d9) as a function of Stress and Load (means and SE) at baseline and 0-, 10- and 20-minutes post-stress.
Time Load TSST SECPT MAT Control
0 Min 1 0.90 (0.14) 0.99 (0.18) 0.91 (0.19) 0.73 (0.19)
2 0.55 (0.14) 0.63 (0.16) 0.42 (0.14) 0.45 (0.14)
320.03 (0.10) 20.08 (0.10) 0.11 (0.12) 20.02 (0.12)
10 Min 1 0.80 (0.18) 0.86 (0.17) 0.89 (0.17) 0.76 (0.19)
2 0.63 (0.15) 0.47 (0.12) 0.49 (0.13) 0.53 (0.14)
320.01 (0.13) 20.04 (0.11) 0.01 (0.10) 20.02 (0.11)
20 Min 1 0.89 (0.18) 0.78 (0.18) 0.88 (0.19) 0.61 (0.21)
2 0.51 (0.16) 0.54 (0.16) 0.50 (0.14) 0.57 (0.14)
3 0.01 (0.13) 0.00 (0.12) 20.14 (0.13) 0.04 (0.10)
No significant effects were found for Stress, Time, Load or any interactions.
doi:10.1371/journal.pone.0113618.t004
Comparison of Three Psychosocial Stress Paradigms
PLOS ONE | DOI:10.1371/journal.pone.0113618 December 12, 2014 11 / 1 9
Fig. 3. Heart rate. Heart rate as a function of stress condition and time (means and standard errors) pre-
stress, during the stressor, and at 0, 10, and 20-minutes post-stress. Across all Stress conditions, heart rate
was higher than baseline during the Stress tasks, and lower than baseline at all time-points post-stress
(p,0.01). Additionally, heart rate higher than baseline during the TSST and lower than baseline 10-30 min
post-TSST (p,0.01). Heart rate was higher than baseline during the SECPT and lower than baseline at all
time-points post-SECPT (p,0.01). Heart rate was lower than baseline during all time points post-Control task
F(4,92) 59.121, p,0.01. Heart rate did not change over time during the MAT.
doi:10.1371/journal.pone.0113618.g003
Fig. 4. Salivary cortisol concentration. Salivary cortisol as a function of stress condition and time (means
and standard errors) pre-stress and at 0-, 10-, and 20-minutes post-stress. Although ANOVAs were performed
on log-transformed data, the figure shows raw data. Cortisol levels were higher than baseline at all time-points
following the TSST (p,0.01), but not the SECPT, MAT or control task (all ps..08). Additionally, cortisol levels
were higher during the TSST and SECPT conditions 20-minutes post-stress (p,0.05) but not during the the
MAT or for any stressors pre-stress, or immediately or 10 minutes post-stress (all ps..06).
doi:10.1371/journal.pone.0113618.g004
Comparison of Three Psychosocial Stress Paradigms
PLOS ONE | DOI:10.1371/journal.pone.0113618 December 12, 2014 12 / 19
Salivary Alpha-Amylase (AA)
Analysis of baseline differences between the four Stress conditions yielded a main
effect of Stress F(3,69) 53.594, p,0.05. sAA was higher before the TSST than
Control (p,.01) and marginally higher before the SECPT than Control (p,0.1).
No effects were found for Stress or Time, nor Stress x Time interactions (Table 5).
Discussion
We compared the magnitude and duration of three experimental psychosocial
stress induction paradigms on physiology, mood, and cognition. As hypothesized,
the Trier Social Stress Test (TSST) exerted the most robust mood and
physiological effects, followed by the Socially Evaluative Cold Pressor Task
(SECPT). Specifically, the TSST increased feelings of tension, total mood
disturbance, salivary cortisol and heart rate as well as reduced feelings of fatigue.
With the exception of heart rate, which declined beginning 10-minutes post-
stress, all TSST effects were evident immediately post-stress as well as 10- and 20-
minutes after stress cessation. The SECPT increased heart rate as well as decreased
feelings of fatigue. Effects of the Mental Arithmetic Task (MAT) were limited to
increasing total mood disturbance. Comparisons of the stress tasks to the control
task showed that relative to the control, the TSST resulted in elevated feelings of
tension immediately post-stress, and both the TSST and SECPT reduced feelings
of fatigue and increased cortisol levels 20-minutes post-stress. The stress response
duration is a key to planning a study that utilizes an experimental stressor, as any
dependent variable must be measured before the stress response subsides. The
TSST would allow a longer window to administer tasks of interest.
There are several explanations as to why the MAT evoked a smaller stress
response than the TSST and SECPT. First, the socioevaluative component of the
TSST and SECPT are thought to contribute to the stress response [10,36,37].
Because the socially evaluative component of the MAT involves viewing another
individuals’ test score relative to one’s own score rather than face-to-face contact
with the evaluator, the MAT may invoke less socially evaluative pressure. Second,
individual differences in arithmetic ability may have left this task susceptible to
heterogeneity of responses. Average (¡SEM) scores on the control MAT task
were 67.36¡2.95 compared to 61.22¡5.84on the stressful MAT, and the average
difference score between the control and stressful task was only 3.22¡3.99. Only
11 out of the 24 subjects showed a decrease in score between the control and
stressful MAT. Previous research has shown the individual differences in math
anxiety influences math performance and cortisol response [38–40]. We did not
find additional effects when we restricted the analyses to individuals whose scores
were lower in the stressful versus control MAT, but this could be due to low
sample size in this group.
The influence of the TSST and SECPT on mood was consistent with previous
studies [15,18,41]. The TSST increased salivary cortisol levels, a biomarker of
HPA activation, and both the TSST and SECPT resulted in higher cortisol levels
Comparison of Three Psychosocial Stress Paradigms
PLOS ONE | DOI:10.1371/journal.pone.0113618 December 12, 2014 13 / 19
than the control task 20-minutes post-stress. Participants were asked to keep their
arms in the water for 3 minutes, but most withdrew their arms earlier (mean ¡
SEM 574.0 ¡21.5 seconds). Although this duration is within the range of
studies that used the cold pressor to assess pain tolerance [42], it is shorter than
that observed in other studies finding cortisol effects of the SECPT [18,19,43] and
may account for the null effects. Of course, obtaining institutional review
approvals for forced submersion durations might prove difficult or impossible.
Future studies using this task should measure pain and perceived stress, to
determine whether pain sensation supersedes the stress effects of this task.
Contrary to our hypotheses, we did not find stress effects on salivary alpha-
amylase (AA), a biomarker of autonomic nervous system activation. Although AA
levels fluctuate throughout the day, with a steep decline within 30 minutes of
waking followed by gradual increase throughout the day [44], this is not likely to
account for our null findings, as all participants began their test sessions in the
afternoon. The majority of previous studies found that experimentally-induced
stress increased AA levels [23,45], but some did not [27].
The literature on stress effects on working memory is mixed. Several
neuroimaging studies assessed the influence of stress on working memory-related
brain activation. Qin and colleagues (2009) found that stress impaired N-Back
working memory performance and reduced working memory-related DLPFC
activity. Conversely, Porcelli et al. (2008) found increased activation in the PFC,
with no corresponding change in working memory performance [46]. A third
study found that stress enhanced working memory performance and increased
activity in the PFC and posterior parietal cortex (PPC) [47].
Task difficulty could account for the differential findings. For example, stress
impaired performance on active tasks that require constant updating of
information, including the OSPAN [18,28] and N-Back [15,48] but not passive
tasks including the digit span [27,29]. Mixed effects were found on the Sternberg
paradigm, in that stress reduced response time but increased false alarms [17].
Alternatively, differences in N-Back stimuli may explain differences in results.
Whereas our task used spatial stimuli (i.e. participants responded whether the
stimulus was in the same location as the stimulus1-, 2-, or 3-back), other studies
used digits [15,48]. A large body of research has investigated whether the neural
networks of working memory are modality-specific. Although the evidence is
Table 5. Salivary alpha amylase (AA) as a function of stress (means and SE) at baseline and 0-, 10- and 20-minutes post-stress.
TSST SECPT MAT Control
Pre-Stress 31.15 (5.04) 30.46 (8.38) 24.34 (4.37) 18.31 (2.64)
0 min 29.64 (4.97) 28.16 (5.23) 33.08 (5.70) 29.16 (4.70)
10 min 28.20 (4.66) 28.07 (6.45) 24.88 (4.10) 25.20 (3.24)
20 min 26.83 (4.39) 27.63 (5.94) 22.97 (3.78) 22.08 (3.74)
Although ANOVAs were performed on log-transformed data, the figure shows raw data. No significant effects were found for Stress or Time, nor Stress x
Time interactions.
doi:10.1371/journal.pone.0113618.t005
Comparison of Three Psychosocial Stress Paradigms
PLOS ONE | DOI:10.1371/journal.pone.0113618 December 12, 2014 14 / 19
mixed, some reviews and meta-analyses report dissociation between spatial and
non-spatial working memory, wherein spatial working memory relies on a dorsal
information stream whereas visual working memory relies on a ventral stream
[49]. However, no differences were found in prefrontal cortex activity between
spatial and non-spatial working memory tasks, making stimuli differences an
unlikely explanation for our failure to replicate stress-induced N-Back working
memory impairment [49,50].
Limitations
A number of limitations apply to the current study, which may at least partially
explain relatively weak findings relative to the stress literature. First, we did not
find changes in AA in response to stress, which is inconsistent with extant findings
[51]. The most likely explanation for such null results is baseline differences in AA
levels between the four Stress conditions, which potentially masked stress effects,
as AA levels were higher in individuals before beginning the TSST (p,.05) and
marginally higher (p,.1) before the SECPT than before the Control task. Further,
it has been suggested that the use of Salivettes, as in the current study, may not
yield AA results as reliable as other methods such as spitting or passive drool [35].
Second, the Control Task was not a perfect control condition for all three
stressors. The TSST, MAT and Control task lasted 20 minutes, whereas the SECPT
had a maximum duration three minutes, and as previously discussed, the majority
of the subjects did not reach this threshold. Thus the Control Task controlled for
the duration of the TSST and MAT but not the SECPT. Further, participants were
required to walk to an adjacent room to complete the TSST and SECPT, whereas
those undergoing the MAT and Control task remained seated. The increased
movement involved in the TSST and SECPT could have contributed to elevations
physiological stress response [36]. In this way, the Control Task controlled for
movement of the MAT but not TSST and SECPT. While acknowledging that the
Control task was not an ideal control for all three Stress tasks, adding a specific
Control task for each stressor would be impractical, in that it would nearly double
the number of test sessions and thus potentiate practice effects.
Third, factors pertaining to participants’ background which could also
influence the stress response were not collected, including experience related to
the tasks, such as public speaking and arithmetic, hours of sleep, and time of
waking prior to testing [52,53]. These variables were not tested in the present
study and thus not included as covariates, potentially contributing to the relatively
high within-group variability.
Finally, our sample of 24 individuals included 7 males and 17 females, meaning
assessing potential gender differences in stress-induces changes to working
memory was infeasible. We feel that our total sample size was sufficient, given that
we employed a repeated measures design, whereas previous studies utilizing larger
samples assessed between-subjects differences between stress and control groups
[15,48,54,55]. Further, our sample size is in line with other studies assessing
stress effects on memory that have used repeated measures designs [46,48,56,57],
Comparison of Three Psychosocial Stress Paradigms
PLOS ONE | DOI:10.1371/journal.pone.0113618 December 12, 2014 15 / 19
even those that have used between measures designs [28]. That said, the small
number of males within the sample precluded any analysis of gender differences.
Cortisol levels following stress were generally higher in males than females [58,59]
and cortisol responses were inversely associated with memory performance in
males but not females [60]. Subsequent studies should utilize a larger sample size,
particularly one large enough to examine potential gender differences.
Conclusions
Comparison of the TSST, SECPT and MAT task indicates that the TSST and
SECPT are likely the most effective methods of experimentally inducing acute
stress, with the TSST proving the most robust and reliable. Together with previous
studies, our data suggests that the TSST and SECPT impairs mood and increases
HPA axis activity. The SECPT and MAT have the logistical advantage of requiring
fewer investigators. Additionally, the MAT would enable investigators to run
multiple subjects at once. However, participants are allowed to withdraw their
arms from the cold water in SECPT when they wish, and many do so soon after it
becomes uncomfortable. Therefore the physiological and mood effects of this task
are generally limited to the duration of the stressor, thus reducing its utility in
research requiring more sustained stress response.
Similarly, individual variability in arithmetic performance, and likely math
anxiety, contributes to inconsistent effects of MAT task. Although the MAT did
impair some aspects of mood, it had no influence on HPA or autonomic activity.
Perhaps limiting participants to those with high math anxiety would increase the
efficacy of the MAT task as an experimental stressor. However the task is not
effective in a random cross-section of young adults. In conclusion, the present
data support the continued use of the TSST as the prototypical experimental
stressor, particularly relative to the SECPT and MAT.
Acknowledgments
We would like to thank Rachel Aronchick, Emily Caplan, Jackie Hayes, Neha
Kumar, Vivien Lim, and Pat Oungpasuk for their careful work in data collection.
Author Contributions
Conceived and designed the experiments: GEG CRM TTB HAT RBK. Performed
the experiments: GEG. Analyzed the data: GEG TTB. Wrote the paper: GEG CRM
TTB HAT RBK.
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