Psychoneuroendocrinology (2011) 36, 11271136

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p s y n e u e n

Assessing stress reactivity indexed via salivary cortisol in preschool-aged children

Katie R. Kryski a,*, Heather J. Smith a, Haroon I. Sheikh b, Shiva M. Singh b, Elizabeth P. Hayden a
a b

Department of Psychology, Westminster Hall, University of Western Ontario, London, ON, Canada N6A 3K7 Department of Biology, Molecular Genetics Unit, Western Science Centre, University of Western Ontario, London, ON, Canada N6A 5B7

Received 23 August 2010; received in revised form 10 January 2011; accepted 8 February 2011

KEYWORDS
HPA; Cortisol; Stress; Children; Multi-level modeling

Summary Identifying a stressor paradigm that elicits mean increases in salivary cortisol in young children has proven elusive, possibly due to characteristics of the paradigms used and how and when cortisol is sampled. We therefore examined the validity of a standardized task (adapted from Lewis and Ramsay, 2002) and procedures developed to assess cortisol reactivity in 215 preschool-aged children. Children participated in a standardized stress task during a home visit, which was videorecorded for future coding. Salivary cortisol samples were obtained at baseline and 10, 20, 30, 40, and 50 min post-stress. In support of the validity of the task, signicant increases in cortisol levels from baseline were found, followed by a signicant decline, and a quadratic function provided a good t to the data. Children also showed a signicant increase in negative emotions and a decrease in positive emotions over the course of the stress task. Results indicate that the task successfully elicited the hypothesized cortisol response in 3-year-old children. # 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Individual differences in stress sensitivity have been strongly implicated in the etiology of major depression and anxiety disorders (Heim and Nemeroff, 2001; McFarlane et al., 2005), suggesting that early-emerging indicators of heightened stress sensitivity could be used to develop targeted

* Corresponding author. Tel.: +1 519 611 2111x80348; fax: +1 519 850 2554. E-mail addresses: kkryski2@uwo.ca, krkryski@gmail.com (K.R. Kryski).

preventative and early intervention strategies. As betting a topic with such important implications, numerous studies have aimed to measure childhood stress reactivity, operationalizing sensitivity to stress via activity of the hypothalamicpituitaryadrenocortical (HPA) axis in response to standardized stress paradigms. Cortisol, the hormonal endproduct of the HPA system, can be readily indexed noninvasively through salivary assays and is a potentially useful tool in developmental psychopathology research aimed at characterizing stress responsivity in childhood. However, the methods used to elicit cortisol reactivity in children vary widely, and the resulting literature is, accordingly, mixed and inconclusive.

0306-4530/$ see front matter # 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2011.02.003

1128 Gunnar et al. (2009) recently provided an excellent review of this literature, highlighting the substantial variability in methodologies used to elicit cortisol in early childhood. In this review, the authors note that only a small minority of studies of young children (i.e., ages 25 years old) report a mean increase in child cortisol in response to standardized stressor paradigms (Gunnar et al., 2009). Gunnar and colleagues concluded that without concerted effort to develop reliable stress tasks for this age range, we may be left with a broad span of development during which we cannot really assess reactivity of the axis in typically developing children (p. 964). Despite the absence of a consistent literature to draw upon, Gunnar et al. (2009) made recommendations regarding task characteristics that may elicit a cortisol increase in young children. For example, Gunnar et al. observed that leading children to believe that they have failed on a task that younger children are capable of doing may elicit a cortisol increase. This suggestion is remarkably consistent with results from a recent meta-analysis of cortisol reactivity in adults (Dickerson and Kemeny, 2004), which showed that tasks that entailed a socially evaluative component, along with perceived uncontrollability and motivated performance, produced the greatest and most prolonged cortisol response. While this meta-analysis included studies of adults only, designing developmentally appropriate downward extensions of tasks that elicit cortisol responses in older samples would represent a valuable contribution to the eld, as research aimed at examining stability and change in cortisol reactivity across development will be facilitated by tasks that are similar in terms of the nature of the stress manipulation. An array of methodological considerations may have inuenced the extent to which previous research has been able to accurately characterize early-emerging cortisol reactivity in young children. The vast majority of studies of cortisol reactivity to laboratory stressors have assessed cortisol at two time points only (Earle et al., 1999; Matthews et al., 2001; Roy et al., 2001; Smeekens et al., 2007; for exceptions, see Mills et al., 2008; Zoccola et al., 2008). This practice may have limited the extent to which studies can accurately capture peaks in cortisol response, as individuals, including children, vary in terms of how rapidly a maximum cortisol response is expressed post-stressor (Goldberg et al., 2003; Gunnar and Talge, 2008; Lewis and Ramsay, 2002; LopezDuran et al., 2009). Additionally, obtaining a minimal number of samples hinders the ability to characterize post-stress downregulation/recovery, which may also have implications for psychopathology risk. Other methodological considerations include where and when assessments of cortisol reactivity are conducted. Many previous studies have assessed cortisol reactivity in children in laboratory settings (Lewis and Ramsay, 2002; Lopez-Duran et al., 2009; Smeekens et al., 2007). While laboratory settings have advantages, this approach raises the concern that the novelty of the setting itself might inuence childrens cortisol levels. Research by Tottenham et al. (2001) indicates that this is so, by showing that assays of children yield higher levels of baseline cortisol when samples are collected in the laboratory versus the home (as cited in Gunnar and Talge, 2008, p. 351). This indicates that the baseline cortisol levels reported in many studies reect a psychophysiological reaction to coming

K.R. Kryski et al. to a laboratory, rather than a true baseline measure of cortisol activity, and suggest that testing done in the home may better identify true increases and recoveries in cortisol levels in response to stressors of interest. Also, cortisol varies throughout the day, with peak levels being reached upon awakening, and lowest levels found after sleep onset (deWeerth et al., 2003; Gunnar and Talge, 2008). This natural variability can introduce additional, nonsubstantive variability into cortisol assays, which is problematic given that most studies of childrens cortisol responses to stress have tested participants at different times of day. With these issues in mind, we examined whether a signicant cortisol increase could be elicited from preschoolaged children using a task adapted from Lewis and Ramsay (2002). This task, which already incorporated many features advocated by Gunnar et al. (2009) for use in tasks to elicit cortisol from children, was further adapted to be suitable for use with younger children, and to incorporate additional elements implicated by Dickerson and Kemeny (2004). Additionally, we attempted to control or eliminate nonsubstantive inuences (e.g., time of day) on childrens cortisol.

2. Methods
2.1. Participants
Participants were an unselected community sample of 215 three-year-old children from southwestern Ontario, who were recruited for a larger study of genetic and other biological and contextual inuences on child temperament and psychopathology risk. Children were recruited by contacting families through a universitys developmental research participant pool and by advertisements placed in local daycares, preschools, recreational facilities, and on websites. Children with signicant medical or psychological problems were excluded from participation via a screening procedure administered by trained study personnel at the recruitment stage. Written consent for child participation was obtained from the primary caregiver, who was told that their child would be asked to do a matching task that was designed to be impossible to nish, and that to minimize distress to the child after the task, s/he would be told that their failure to nish was due to malfunctioning equipment (see Section 2.3 for further details on the task). Participating families identied themselves as Caucasian (N = 195; 90.7%), Asian American (N = 5; 2.3%), or other race (N = 15; 7.0%) Over half of families (54.7%) reported a family income ranging from $40,000 to $100,000 CAD; 27.9% of families reported a family income greater than $100,001 CAD; 10.9% of families reported a family income ranging from $20,000 to $40,000 CAD; and 6.5% of families reported a family income of less than $20,000 CAD. Approximately half of the mothers (52.4%) and fathers (53.3%) either graduated from high school (or received a GED), attended some college, or received a 2-year degree as their highest level of educational attainment; 44.3% of mothers and 38.2% fathers received a 4year college degree or beyond; and 1.4% of mothers and 2.9% of fathers had completed some high school. The mean age of parents was 32.9 years (SD = 4.9) for mothers and 34.7 years (SD = 4.7) for fathers. The mean age of child participants was 3.5 years (SD = .3), and 53.5% were female. The majority (86.6%) of the children came from two-parent homes. The

Assessing stress via salivary cortisol family demographic data for this sample closely resembles the most recent London, Ontario census data available (Statistics Canada, 2006). Children were administered the Peabody Picture Vocabulary Test (PPVT; Dunn and Dunn, 1997) to screen for gross cognitive impairment and English prociency (M = 112.41, SD = 14.81).

1129 At the beginning of the task, the child was allowed to choose a prize from an assortment of small toys. The child was then told that each bear on the board should get a blue ball, and each frog a red ball (the balls being game pieces with adhesive backing). To ensure comprehension of the task, the child was given several opportunities to practice matching the animals with the correct color game piece prior to starting the task. The child was then told that the trafc light would show how much time they had to match the animals with the correctly colored balls. More specically, as the experimenter used the control to change the color of the trafc light, the child was told that they had plenty of time to work when the light was green, but that when the light turned yellow, they were running out of time, and that when the light turned red, they were out of time. The red light was accompanied by a loud buzzer sound. Children were told that they must match all the animals on the board with the right ball to get their preferred prize (the previously selected toy). Consistent with Gunnar et al.s (2009) recommendations, to further enhance the stress-inducing nature of the task, children were told that the matching task was easy to do, and that even little kids could nish on time. The child was then told to begin the matching task. For most children,2 the green light on the stop light shined for 2 min and 20 s. The experimenter then surreptitiously changed the signal to the yellow light via the remote control, and exclaimed that the child was running out of time. After another 40 s, the experimenter switched the light to red, which triggered the loud buzzer. At this time, the experimenter told the child that they did not nish in time and that they would not get the preferred toy. Two subsequent, identical trials occurred in which children were again unsuccessful at nishing the task. The experimenter recorded how many pieces the child placed correctly and incorrectly for each consecutive trial. Upon the conclusion of the third trial, the experimenter exclaimed that the light was broken and that the child had not been given enough time to nish the task. The childs matching skills were then praised and the child received their preferred toy. Following the conclusion of the task, children were allowed to interact with their parent if they desired to do so. Additional parentchild contact occurred during a free play task that followed the cortisol sampling, during which the caregiver and child played with a set of novel, pleasant toys. The mean duration of the task for children who completed all three trials was 15.01 min (SD = 1.54), including the instruction period. This task length is consistent with most tasks used in the literature (Dickerson and Kemeny, 2004). Due to extreme negative reactions or refusals to continue by some children (N = 26, 12.5%), all three trials of the task could not be completed. As task length varied somewhat between children, it was used as an independent variable in analyses. However, task duration has no known inuence on

2.2. Procedure
Testing was done in childrens homes by a female experimenter, whom the children knew from previous study procedures, to reduce extraneous increases in cortisol. All home visits began between 12:00 pm and 3:30 pm in the afternoon to minimize the effects of diurnal variation on cortisol. Parents were instructed not to allow their child to eat or drink anything other than water for one half hour prior to the visit to minimize error in cortisol assays (Magnano et al., 1989; Schwartz et al., 1998). At the beginning of the home visit, the child and experimenter played together quietly with toys (e.g., books, coloring, blocks) for 30 min to allow any increases in childrens salivary cortisol due to the arrival of study personnel to decrease before baseline samples were taken. During this time, the child was encouraged to stay seated and engage in minimal activity, as cortisol levels are also inuenced by physical activity (Wellhoener et al., 2004). After 30 min had passed, a baseline salivary cortisol sample was collected, followed by the stress task described below. Following the stress task, the child and experimenter again resumed quiet play while the remaining cortisol samples were collected at 10, 20, 30, 40, and 50 min post-stressor. Consistent with the goal of minimizing stress to children before and after the task, children could interact with their parent during the warm-up period and after the stress task, but typically preferred to engage in play with the experimenter. The stress task was videotaped by a female research assistant for subsequent coding.1

2.3. Stress task

Each child participated in a stress task adapted from previous work by Lewis and Ramsay (2002) in which 4-year-old children matched colored stickers to animals on a worksheet using a key. Pilot data indicated that the task was too difcult for 3year-old children to understand in its original form. Therefore, the task was simplied to make it developmentally appropriate for 3-year-olds, and was further modied to include a greater number of features thought to increase cortisol reactivity (Dickerson and Kemeny, 2004; Gunnar et al., 2009). For the task, each child and the experimenter were seated beside each other at a table in front of a large felt board on which numerous bear and frog icons had been afxed. A large toy replica of a trafc stoplight (a YackerTracker1, modied for the present study) was placed adjacent to the board, and the experimenter used an unobtrusive remote control to manipulate the trafc light. Prior to beginning the task, parents were asked to unobtrusively observe from outside the childs sight if they wished to be present during study procedures.

The full study protocol is available by request.

As some children were very good at the task, time was adjusted at the discretion of the experimenter to ensure that all children were unsuccessful at completing the task. Such adjustments were not usually needed as the times used for the task were established using pilot data, which indicated that most children would not be able to complete the task in 3 min. 3 Available from rst author.

1130 cortisol reactivity and longer tasks do not elicit stronger responses, even when comparing tasks ranging from 3 to 60 min in duration (Dickerson and Kemeny, 2004) and excluding the children who failed to complete all three trials from analyses yielded virtually identical results.3

K.R. Kryski et al. decreased as a result of the stressor. As a baseline measure of child PE and NE, relevant child behaviors during the experimenters introduction of the task, until the point where children were shown the red light and told that they could run out of time, were coded.5 Changes in PE and NE expressed at baseline relative to the stressful portion of the task were calculated by subtracting summed instances of emotion expressed during baseline from that expressed during the stressful portion of the task after rst controlling for the length of the two intervals. Childrens activity level during the task was also coded, as activity level can produce changes in cortisol (Wellhoener et al., 2004). Activity level was rated using a global rating scale ranging from 0 to 3, written such that a score of 0 reected lower than typical activity, midrange scores reected typical levels of activity, and scores of 3 reected greater than typical activity level. Videos were coded by trained graduate and undergraduate students, and 15% of the coded videos were coded by a master coder for reliability. The average intraclass correlations for NE, PE, and activity level ratings were good (.91, .96, and .83 respectively).

2.4. Cortisol sampling procedure

Cortisol samples were obtained at baseline (immediately before the introduction of the stress task), and at 10, 20, 30, 40, and 50 min following the end of the matching task. To collect saliva, children chewed on an absorbent cotton dental roll until it was wet. To facilitate compliance, sampling was presented as a game in which the child raced the main experimenter to get a few grains of red Kool-Aid1 out of a colorful Dixie1 cup, receiving stickers upon completion of each sample. Kool-Aid1 was used sparingly, and previous work shows that its use does not signicantly alter the pH of the saliva (Talge et al., 2005). After each sample was obtained, a research assistant expunged the saliva into a labeled micro tube, and all samples were frozen immediately upon return to the laboratory following the visit. Samples were later taken to a laboratory at the University of Western Ontario where they were assayed in duplicate using an expanded range, high sensitivity, salivary cortisol enzyme immunoassay kit (Salimetrics, PA, USA). Optical density was read on a standard plate reader at 450 nm and corrected at 650 nm (Molecular Devices, Sunnyvale, CA, USA). All samples from the same child were assayed in the same batch with no duplicates varying more than 5%. Enzyme immunoassays were run according to manufacturer instructions and average intra- and interassay coefcients were 3.5 and 5.1% respectively. Values from matched serum and saliva samples show the expected strong linear relationship, r(47) = .91, p < .0001 (Aardal and Holm, 1995; Dorn et al., 2007). Standard curve and concentration of unknown samples was generated according to manufacturers instructions using a 4parameter sigmoid minus curve t (SPSS 16.0). Non-compliance rates for samples collected from young children generally range from 8 to 10% (Lewis and Ramsay, 2002; Mills et al., 2008; Blair et al., 2008). Two hundred and six children provided all six cortisol samples (95.8%) while the remaining nine children (4.2%) did not provide a sample at least one time point.4 Non-compliance rates based on the number of overall samples collected was 18 samples out of 1290 attempted (1.4%).

3. Results
3.1. Data analysis techniques
As is typically found (Gunnar and Talge, 2008), our cortisol values were positively skewed. A log 10 transformation of the raw cortisol values yielded unskewed cortisol values that were used in all analyses. Table 1 presents correlations between mean cortisol levels at each sampling time and all major study variables. All correlations involving NE and PE were partial correlations controlling for baseline NE and PE values. As expected (Wellhoener et al., 2004), cortisol at 10, 20, 30, 40, and 50 min post-stress was positively correlated with child activity. Cortisol levels 10 min post-stress were negatively associated with child age. Thirty-, forty-, and fty-min cortisol levels were associated with NE during the task. NE during the task was correlated with both PE and activity level during the task. Task length was negatively correlated with PE and NE during the task, and was correlated with child sex such that the task duration was shorter for females than males (although this difference was marginal; Mgirls = 12.0 min, SD = 2.3, Mboys = 12.6 min, SD = 1.5).6 Activity level was associated with time of day and child age; older children and children tested later in the afternoon were more active.

2.5. Child behavior coding

In order to conrm that children experienced the stress task as stressful, facial, verbal, and physical displays of childrens positive and negative emotions (PE and NE, respectively) were coded to examine whether NE increased and/or PE

4 One child refused to provide a 50 min sample; two children did not provide 40 and 50 min samples; ve children failed to provide 10 and 20 min samples; and one child refused to provide 10, 20, and 50 min samples. 5 Pilot data suggested that this juncture of the task was when most children began to perceive the task as stressful.

The association between child sex and task length appears to be due to the fact that girls were generally better at the matching task than boys; girls placed signicantly more pieces correctly (trial 2: (t(171) = 2.51; trial 3: (t(171) = 2.491) and made signicantly fewer errors (trial 2: (t(171) = 3.36; trial 3: (t(171) = 3.38) than males ( ps < .05). However, considering that the task was designed so that all children failed, regardless of skill, and that there were no sex differences obtained for cortisol levels or child affect during the task, the sex difference in task length (or in the ability to do the task) appears not to have inuenced the extent to which male and female children perceived the task as stressful.

Assessing stress via salivary cortisol

Table 1

Correlations among variables. 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1. Baseline cortisol 2. 10 min .62 ** .83 ** 3. 20 min .56 ** ** .64 ** .77 ** 4. 30 min .56 ** ** 5. 40 min .57 .53 .60 ** .80 ** .43 ** .50 ** .60 ** .79 ** 6. 50 min .50 ** 7. PE during task .03 .02 .06 .01 .02 .12 .18 * .15 * .18 * 8. NE during task .07 .04 .11 .24 ** ** ** ** ** * 9. Activity level .10 .20 .24 .26 .23 .17 .21 * .15 * .15 * .08 10. Length of task .01 .05 .06 .11 .09 .00 .21 * .03 11. Time of day .06 .08 .09 .13 .12 .06 .08 .02 .17 * .09 .08 .10 .12 .01 .03 .14 * .07 .01 12. Child age .03 .14 * 13. Child sex .06 .08 .08 .02 .02 .08 .02 .00 .05 .17 * .05 .04 14. PPVT .03 .05 .03 .05 .05 .04 .06 .10 .011 .10 .05 .03 .11 Mean (SD) 1.28 (.25) 1.08 (.31) 1.04 (.34) 1.04 (.32) 1.09 (.32) 1.16 (.35) 1.91 (1.78) 2.97 (1.99) 1.5 (.9) 15.01 (1.5) 2:00 (1.25h) 3.5 (.31) 112.41 (14.81)
Note: Cortisol levels are measured in microgram per deciliter (mg/dl). All correlations involving NE and PE were partial correlations (controlling for baseline NE and PE values). Child sex: male = 1 and female = 2; h = hours. * p < .05. ** p < .01.

1131

1132

K.R. Kryski et al.

3.2. Child affect during the task

Indicating that the task was indeed perceived as stressful by children, paired t-tests showed a signicant decrease in PE from baseline compared to the stressful portion of the task, and a signicant increase in NE from baseline compared to the stressful portion of the task, t(215) = 3.68 and t(215) = 13.95, p < .001, respectively. No signicant sex differences were found for NE or PE difference scores or for NE and PE expressed during baseline or during the stressful portion of the task ( ps > .42).

3.3. Cortisol level comparisons across sample

Paired samples t-tests were used to test differences between mean cortisol levels across time. Mean cortisol levels increased signicantly from baseline to 10 min, t(207) = 11.09; p < .001, and from 10 to 20 min, t(207) = 3.40, p = .001, post-stress. There was no signicant change from 20 to 30 min post-stress, p = .61 (see Table 2). Mean cortisol levels then began to decrease and signicant reductions were found in mean levels from 30 to 40 min, t(211) = 2.95, p < .05, and 4050 min, t(209) = 4.13, p < .001, post-stress (see Table 1 for mean cortisol values). The average cortisol trajectory of log 10 transformed data can be seen in Fig. 1. To examine overall cortisol trajectories for the sample and predictors of individual differences in these trajectories we used multi-level modeling (MLM) conducted with HLM 6 (Scientic Software International Inc., IL). MLM has a number of advantages, such as allowing data to be modeled at two levels (Level 1, describing within-individual change over time; and Level 2, relating predictors to any interindividual differences in change), and accounting for missing values at Level 1 (Singer and Willett, 2003). For the Level 1 model, cortisol time points (baseline, 10, 20, 30, 40, and 50 min) were nested in the Level 2 variable, participant. Log 10 transformed cortisol values were the dependent variable. To evaluate the model, the following function was specied to describe the data from each individual:
Table 2 Effect Baseline to Baseline to Baseline to Baseline to Baseline to 1020 min 1030 min 1040 min 1050 min 2030 min 2040 min 2050 min 3040 min 3050 min 4050 min
* **

Figure 1 Mean child cortisol level (mg/dl) as a function of cortisol sampling time. Figure displays data that has been log 10 transformed.

Level 1 Level 2

Y i j b0 j b1 j Time b2 j Time2 ri j Intercept : b0 j g 00 u0 j Instantaneous rate of change : b1 j g 10 u1 j Curvature : b2 j g 20 u2 j

(1)
where Yij is cortisol values of individual j at time i; b0j is the cortisol value of individual j at Time = 0 (i.e., the baseline cortisol value of individual j); b1j is the instantaneous rate of the linear change in cortisol for individual j at Time = 02; b2j is the rate of the curvature in cortisol; and rij is the residual variance in repeated measurements for individual j. Between-subjects predictors of individual change were also modeled to allow examination of cortisol levels at each sampling time across participants, or for each individual, while taking into account between persons predictors. For these analyses, Level 2 predictors were activity level, time of day, length of task, child sex, child age, and child NE and PE during the task after controlling for baseline levels. We also examined whether any of the between-subject variables interacted to predict childrens HPA axis reactivity. Time was anchored at baseline (time = 0) so that the cortisol intercepts (b00) would reect the average of individuals cortisol levels at baseline. All Level 2 between-person variables were centered at their grand mean. MLM is equipped to handle missing data at Level 1 by estimating the trajectory based on existing data for that participant. Individuals with data missing at Level 2 were excluded during analysis (n = 4). A quadratic equation was built to examine the effects of Level 2 variables on the intercept, instantaneous rate of change (henceforth referred to as slope), and curvature (Eq. (1)). Conrming the selection of a quadratic model, a chi-square test of the deviance statistics between unconditional linear and quadratic models indicated that adding a quadratic term to the model resulted in a signicant improvement in model t ( p < .05) (see Table 3), supporting results from simple t-tests indicating that most children showed an initial increase in cortisol from baseline followed by a decrease.

Changes in cortisol over time using paired t-tests. M 10 min 20 min 30 min 40 min 50 min .19 .24 .24 .19 .12 .05 .04 .01 .07 .01 .05 .12 .04 .11 .07 SD .26 .29 .28 .29 .32 .20 .27 .32 .37 .23 .31 .36 .21 .31 .23 SE .02 .02 .02 .02 .02 .01 .02 .02 .03 .02 .02 .02 .01 .02 .02 t-Value 11.09 ** 11.89 ** 12.12 ** 9.89 ** 5.57 ** 3.40 ** 2.05 * .25 2.81 * .51 2.39 * 4.76 ** 2.95 * 5.07 ** 4.13 **

p .001. p < .05.

Assessing stress via salivary cortisol

Table 3 Comparison of tting no change, linear, and quadratic change trajectories to cortisol data. Parameter Fixed effects Composite model Intercept TIME (linear term) TIME2 (quadratic term) Variance components Variance in intercept Variance in slope Variance in curvature Goodness-of-t Deviance statistic Chi square test value for df = 2
n = 215. * <.05. ** <.001.

1133

No change

Linear change

Quadratic change

g00 g10 g20 s2 0 s2 1 s2 2

60.02 **

56.02 ** 5.04 **

57.87 ** 14.19 ** 13.19 ** .054 * .000 .000 98.66 143.62 **

.065 **

.062 ** .000 **

251.53

242.28 9.25 *

Next, we examined the main effects of NE and PE during the task on the intercept, slope, and curvature for individuals cortisol trajectories after controlling for baseline levels of the respective emotion by entering them simultaneously into the Level 2 equation. First, the univariate effects of these variables were examined in a quadratic growth model (see Table 4). PE during the stressful portion of the task was signicantly associated with slope and curvature. The slope coefcient indicated that for every unit increase in PE there was a .002 decrease in slope and a .00004 increase in curvature ( p < .05), such that greater PE displayed during the task was associated with a less linear, more quadratic trajectory. Activity level was signicantly

associated with both slope and curvature, such that a one unit increase in activity level was associated with an .004 slope ( p < .01) increase and a .00006 decrease in quadratic curvature ( p < .05), showing that children who were more active during the task had a slower return to baseline values following stress. Length of task was signicantly associated with slope such that a unit increase in length of task was associated with a .001 decrease in slope ( p < .05) indicating that children who performed the task longer had smaller linear increase in cortisol following baseline. Lastly, time of day was signicantly associated with slope ( p < .01) and curvature ( p < .05) such that a unit increase in time of day (task was performed later in the

Table 4 HLM exploratory univariate analyses: childrens cortisol reactivity predicted from child sex, child age, global NE and PE during task, activity level, length of task, demographic variables, and time of day in a quadratic model. Variable Coefcient intercept coefcient (SE) .004 .008 .032 .005 .012 .049 .062 .013 .043 .009 .001 .001 .053 .001 .001 .001 (.013) (.015) (.023) (.011) (.017) (.044) (.070) (.019) (.030) (.024) (.022) (.001) (.064) (.001) (.005) (.005) t-Value Cortisol instantaneous rate of change coefcient (SD) .001 .002 .004 .001 .003 .001 .002 .000 .000 .000 .001 .000 .001 .000 .000 .000 (.001) (.001) (.001) (.001) (.001) (.002) (.004) (.002) (.002) (.002) (.002) (.000) (.006) (.000) (.000) (.000) t-Value Cortisol slope coefcient (SD) t-Value

NE control baseline PE control baseline Activity level Length of task Time of day Child sex Child age Family income Race Maternal education Paternal education PPVT Marital status Maternal work hours Age mom Age dad

.336 .533 1.399 .448 .682 1.126 .884 .676 1.40 .362 .126 .688 .819 1.070 .399 .245

1.720 2.014 * 2.778 ** 2.184 * 2.906 ** .233 .420 .465 .024 .116 .362 .095 .215 .494 .940 1.522

.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000

(.000) (.000) (.000) (.000) (.000) (.000) (.000) (.000) (.000) (.000) (.000) (.000) (.000) (.000) (.000) (.000)

.863 2.489 * 2.312 * 2.077 2.437 * .246 .118 .401 .105 .096 .051 .332 .252 .312 .964 1.279

Child sex: male = 1; female = 2; Race: white = 1; other = 2. * <.05. ** <.01.

1134 day) was associated with a .003 increase in cortisol slope and a .00005 decrease in quadratic curvature. Demographic variables were unrelated to child cortisol reactivity and recovery (see Table 4).

K.R. Kryski et al. baseline cortisol levels may have been obtained, thus increasing estimates of reactivity. In support of this notion, the baseline levels in the present study are comparable to normative levels established during in-home sampling in research examining children age 410 years of age (McCarthy et al., 2009). Also, we minimized the effects of diurnal variation on cortisol by running all participants between 12:00 and 3:30 pm, which should have further decreased nonsubstantive variability in our assays. We incorporated many other elements of tasks known to elicit cortisol increases in adults that have not been used systematically in research on children. Hence, by incorporating multiple elements thought to lead to an elevation in cortisol, our procedures may have been more effective at eliciting a cortisol increase than those used in previous studies. We expected that this stress task would increase childrens negative emotions and decrease positive emotions, relative to baseline. Findings were consistent with this expectation; children showed signicantly more negative emotionality during the task than at baseline, and also exhibited a signicant decrease in positive emotionality. Furthermore, childrens emotion expression was meaningfully related to patterns of cortisol reactivity; PE expressed during the stressful portion of the task was signicantly associated with slope and curvature of the quadratic model when controlling for baseline levels, such that higher PE during the task predicted a lower slope and a signicantly higher rate of quadratic growth than lower levels of PE during the task. In other words, children who displayed more positive emotions during the stressful portion of the task displayed less linear growth and a more rapid return to baseline levels than children with lower levels of PE during the stressor. While speculative, this may suggest that greater PE helps children adapt more effectively to a mild social evaluative threat and results in a smaller increase in cortisol following stress and a more rapid return to baseline. This is consistent with literature suggesting that positive emotions provide a buffer in response to stress (Fredrickson et al., 2003; Wichers et al., 2008). Consistent with the well-established inuence of activity level on cortisol (Wellhoener et al., 2004), activity during the task was signicantly correlated with all cortisol levels at all post-stress time points and signicantly predicted cortisol slope and curvature, indicating that children who were more active during the task had a greater slope and slower rate of quadratic curvature. Although we attempted to minimize childrens activity during the task by having them remain seated throughout, this effect was still found. While it is unclear what additional steps we might have taken to minimize childrens activity levels, it is important to note that the fact that we coded activity level is a strength of this study, as it allowed us to examine its inuence on cortisol. The effect of activity level on childrens cortisol is not typically reported in research assessing child cortisol reactivity even when studies have information on child activity level available (Blair et al., 2008; Lopez-Duran et al., 2009; Lewis and Ramsay, 2002). While not all published studies include tests of the effect of childrens activity on cortisol, considering that wide variations in activity level are common in this age group (Hinkley et al., 2008; Tucker, 2008) and are known to inuence cortisol, this may be important information for future studies to include.

3.4. Interactions between Level 2 predictors

For these analyses, interaction terms were made by rst centering continuous variables, then multiplying two terms to create a term reecting the product of both Level 2 predictors (Aiken and West, 1991). The grand mean centered Level 2 predictor variables were entered in the model followed by the interaction term. Only one signicant interaction emerged: activity level and child age interacted to predict both intercept, instantaneous rate of change, and curvature ( p < .05). To interpret this interaction, child age was recentered at values 1 SD above the mean (3.45) and 1 SD below the mean and a new interaction term was created. The model was rerun allowing activity level to predict intercept, slope, and curvature. Activity level signicantly predicted slope, and curvature for children whose ages were 1 SD above the mean but not for those whose ages were 1 SD below the mean ( p > .45), suggesting that, for older children, higher activity level during the task predicted a higher slope and slower rate of curvature (slope: unstandardized coefcient = .005, SE = .002, t = 2.990, df = 160, p = .003; and curvature: unstandardized coefcient = .000, SE = .000, t = 2.716, df = 160, p = .007).

4. Discussion
Methodological issues may have hampered research on cortisol reactivity in young children, such that few studies have reported the expected pattern of reactivity in childrens cortisol responses to a standardized stress paradigm. In other words, it is unusual to nd a quadratic function in such data, despite the fact that this is what would be expected given the nature of the physiological stress response (Earle et al., 1999; Roy et al., 2001; Smeekens et al., 2007). Using the task and procedures described, we found a signicant increase in childrens cortisol levels from baseline to 10 and 1020 min poststressor, accompanied by a signicant decrease in cortisol levels from 30 to 40 and 40 to 50 min post-stress. Furthermore, results using MLM indicated that a quadratic function was a good t to the cortisol data. Our methods may provide a useful tool for the investigation of childrens psychophysiological responses to stress indexed via cortisol reactivity. The average untransformed salivary cortisol increase from baseline to 20 min post-stress was .07 mg/dl, which is high relative to values reported in the literature. For example, in a study using the task on which ours was based (Lewis and Ramsay, 2002), researchers reported a decrease in the cortisol levels of 60 four-year-old children from baseline to 20 min post-stressor, even though childrens baseline levels were higher than those in the present study. Gunnar and Talge (2008) have shown that samples obtained at laboratory arrival do not match samples obtained at home at the same time of day as the laboratory visit, suggesting that samples obtained in the laboratory may reect the psychophysiological response to coming to the laboratory. By conducting our assessments in the home, a more accurate index of childrens

Assessing stress via salivary cortisol The purpose of this study was to validate an age-appropriate task and set of procedures for the induction of stress in 3-year-old children. Analyses examining change in cortisol over time and childrens emotional responses to the task indicate that this task and procedures were successful in eliciting hormonal and emotional indicators of stress. As this task maps well onto paradigms that successfully elicit cortisol in adults (Dickerson and Kemeny, 2004), and could be readily adapted for use with older children, this paradigm and procedures may aid in the examination of continuity and discontinuity in psychophysiological measures of stress reactivity across development.

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Acknowledgements
The authors would like to thank Margaret Sullivan, Michael Lewis, and Nestor Lopez-Duran for their assistance in the development of the task and procedures, Robert Gardner and Lea Dougherty for their advice regarding data analysis, and Jim Staples for providing access to laboratory equipment.

References
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5. Limitations and future directions

As we have noted, research aiming to elicit cortisol increases in children this age has met with limited success. Thus, our task and methods incorporated a wide array of features aimed at maximizing the likelihood that we would obtain a cortisol increase. We appear to have been successful in obtaining such an increase; however, a limitation of our ndings is that it is unclear whether specic aspects of our paradigm played a more critical role than others in producing this response. Future work could pinpoint which features are most important by dismantling the task into its components. Incorporating a control group that utilized the matching task alone without the added components of negative social evaluation, uncontrollability, and motivation would also be benecial. Along similar lines, we incorporated 30 min of quiet play with the experimenter in order to address any increase in childrens cortisol that might have occurred due to the arrival of the experimenter in the home, and to allow the experimenter to build rapport with the child. We cannot say whether this 30-min delay was necessary as we did not attempt to collect a sample upon immediate arrival at the home, due to the concern that many children would react negatively to such procedures at the very onset of the visit, thus comprising the rest of the assessment procedures. Considering that several well-conducted reviews indicate that a warm-up period such as ours may be essential to obtaining a true baseline and to minimizing noncompliance (Gunnar and Talge, 2008; Jessop and TurnerCobb, 2008), we feel that this was a prudent decision. A nal limitation is that it is unclear whether this task can be used with children younger than 3 years of age, or with clinical samples of children. Research into the tasks effectiveness with these groups would prove useful.

Role of the funding sources

This research was supported by grants from the Canadian Institutes of Health Research (CIHR MOP86458), the Childrens Health Research Institute (CHRI), and the Ontario Ministry of Research and Innovation. These funding agencies had no role in the study design, collection, analysis and interpretation of data, the writing of the report or in the decision to submit the paper for publication.

Conict of interest
None declared.

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