Salivary Cortisol Level, Salivary Flow Rate, and Masticatory Muscle Activity in Response to Acute...

Experimental Section

Gerontology 2004;50:383–392DOI: 10.1159/000080176

Salivary Cortisol Level, Salivary Flow Rate, andMasticatory Muscle Activity in Response toAcute Mental Stress: A Comparison betweenAged and Young Women

Merete Bakkea Anette Tuxena Carsten Eckhart Thomsena Allan Bardowa

Tine Alkjærb Bente Rona Jensenb

aDepartment of Oral Medicine, Clinical Oral Physiology, Oral Pathology and Anatomy, School of Dentistry, andbInstitutes of Exercise and Sport Sciences and Medical Anatomy, University of Copenhagen, Copenhagen, Denmark

Received: February 4, 2004Accepted: March 3, 2004

Merete BakkeDepartment of Oral Medicine, Clinical Oral Physiology, Oral Pathology and AnatomySchool of Dentistry, Faculty of Health Sciences, University of Copenhagen20 Nørre Allé, DK–2200 Copenhagen N (Denmark)Tel. +45 35326554, Fax +45 35326569, E-Mail [email protected]

ABCFax + 41 61 306 12 34E-Mail [email protected]

© 2004 S. Karger AG, Basel0304–324X/04/0506–0383$21.00/0

Accessible online at:www.karger.com/ger

Key WordsElectromyography, mental stress W Masticatory muscle W

Mental stress W Salivary cortisol W Saliva flow rate

AbstractBackground: Age-related effects on physiological stressreactions regarding changes in salivary cortisol concen-tration, saliva flow rate, and masticatory muscle activity,as well as the corresponding perceived mental stressand performance in response to acute stressors, havenot yet been fully described. Objective: Thus, the aim ofthis study was to assess the age-related variations inthese variables in response to minor acute and naturalis-tic stressors in terms of computer tasks. Methods: 13aged (60–70 years old) and 13 young (20–30 years old)women with frequent practice and long experience withcomputer use were recruited by personal contact andflyers. The subjects were healthy and had full dentalarches and no orofacial pain. The computer tasks wererandomized and comprised a mentally demanding, mod-ified Stroop colour-word test (CWT) and a less demand-ing reference test, both with a duration of 20 min andwith equal physical demand. Visual analogue scales forglobal assessment of mental stress and perceived taskdifficulty and performance, measurements of saliva flow

rate and cortisol concentration (unstimulated whole sali-va), as well as surface electromyography of the tempo-ralis and masseter muscles were used for assessment,and Spearman correlation analysis and Anova with re-peated measures were used for statistical evaluation.Results: The perceived task-related stress and task diffi-culty were significantly higher and the personal satisfac-tion with the task performance significantly poorer in theaged women. The cortisol concentration, indicating thestress level, showed a small, but significant increase inresponse to the tasks. Also the saliva flow rate increased.This response was most pronounced in the aged andduring the CWT. The average electromyogram variedsignificantly between age groups and tasks, with higherlevels and shorter relative periods with gaps in the agedwomen and in the CWT. In addition, the peak activity ofthe jaw elevator muscles at mouse clicking was signifi-cantly elevated as a form of co-activation or attention-related activity. Conclusions: The study showed markeddifferences in the response to mental demands in agedas compared with young women. The mental stress,reflected by increases of salivary cortisol concentration,saliva flow rate, visual analogue scale ratings, and activi-ty level of the jaw elevator muscles, was more pro-nounced in the aged women in response to the computertasks.

Copyright © 2004 S. Karger AG, Basel

384 Gerontology 2004;50:383–392 Bakke/Tuxen/Thomsen/Bardow/Alkjær/Jensen

Introduction

Activation of the hypothalamic-pituitary-adrenal sys-tem and the subsequent release of the steroid hormonecortisol from the outer cortex of the adrenal gland are con-sidered major components of the physiological stressresponse in humans [22]. Perceived mental stress in termsof current hassles or problems and anticipation of stress-ful events have been associated with an increase of thesalivary cortisol concentration [29], with increased mus-cle activity [19, 20, 25, 26, 36], as well as with bothreduced [5, 11, 24] and increased saliva flow rates [11].

The use of the plasma and/or salivary cortisol concen-trations is widespread as an index of the activity of thehypothalamic-pituitary-adrenal axis and, therefore, emo-tional stress [33]. The concentrations of free plasma corti-sol and salivary cortisol correlate highly with a ratio of 3:2[35]. Cortisol enters the saliva by passive diffusion [35],and the time lag between changes in plasma and salivarycortisol levels is very short, i.e., 1–2 min [18]. Moreover,saliva samples, in contrast to blood samples, can be col-lected in a non-stressful manner, and the saliva flow ratehas no impact on the salivary cortisol concentration [16].Under basal conditions, cortisol has a characteristic circa-dian secretion rhythm, beginning with a morning maxi-mum 30–50 min after awakening and a gradual declinethroughout the day [8] with a short half-life of !1 h [18].In elderly humans, the circadian variation of the cortisolsecretion has been shown to diminish [7, 32], while thestress responses have been reported to be augmented [12].Higher muscle activity levels have also been observed inthe elderly during manually and mentally demandingtasks requiring high precision and speed, probably as aresult of ageing and a slowed processing as compared withyounger adults [17] as well as the decline in motor controlassociated with ageing [19].

However, no study has examined age-related varia-tions in stress and other mental aspects together with awide range of physiological effects of acute and naturalis-tic stressors such as the mentally demanding Stroopcolour-word test (CWT). We hypothesized (1) that indi-vidual variations in task performance would be reflectedin individual variations in the mental (perceived) as wellas the physiological (cortisol concentration, salivary flowrate, and masticatory muscle activity) stress levels, (2)that similar variations in the stress levels could be ob-tained on a group basis comparing aged and young sub-jects, and (3) that the physiological stress parameters (cor-tisol concentration, salivary flow rate, and masticatorymuscle activity) would be mutually interrelated. Thus the

aims of the present study were to assess and compare per-ceived stress, task difficulty, and performance with thephysiological effects in terms of salivary cortisol, salivaryflow rate, and masticatory muscle activity associated withthe tasks on (1) an individual level and (2) on a group levelcomparing healthy, well-educated aged and young sub-jects and (3) to assess the relationship between the physio-logical effects of the tasks.

Subjects and Methods

SubjectsThe study group comprised 26 healthy Caucasian, unpaid volun-

teers (table 1). As the gender might influence the outcome measures,no men were included [29]. The subjects were 13 aged (60–70 yearsold) and 13 young (20–30 years old) female staff members and stu-dents at the University of Copenhagen or subjects with a similar edu-cational background, recruited through personal contact and flyers.All the subjects had Danish as their first language and gave theirinformed consent to participate in the experiments that were ap-proved by the local ethics committee.

The aged and young women had equally frequent practice (73%used computer daily and the remaining 27% several days per week)in two or more programs on a computer with a computer mouse.Both groups had used computer for several years, although the agedwomen had significantly longer experience (table 1). None of the sub-jects showed dyslexic difficulties. They were all right-handed andmoderately physically active (e.g., cycling, swimming, walking, run-ning, or other exercises for 2–4 h/week on average), and their bodymass index was normal (table 1). They had no history of depressionor neuromuscular or motor system disorders and no other healthproblems that could affect their salivary flow or their jaw elevatoractivity. Five of the subjects took daily prescribed medicine, eitherhormones for birth control and menopausal problems or non-steroi-dal anti-inflammatory drugs for osteoarthritis of the knees. Further-more, the participants did not suffer from temporomandibular disor-ders, frequent headaches, or masticatory muscle pain. They all had atleast 24 teeth or fixed prostheses in full dental arches with posteriorocclusal support.

ProtocolThe subjects were instructed on the day of the investigation to

have a proper breakfast, including milk, juice, or water, and torefrain from eating, drinking, smoking, and performing oral hygieneprocedures the last hour up to the arrival at our laboratory at 09.00 h.No chewing gums or sweets were allowed during the experiments.The experimental session started with a medical interview and a clin-ical examination, including an initial saliva collection at about10.00 h. Then the subject was offered a glass of water, the electrodeswere placed, and maximum jaw elevator activity and bite force wererecorded. The intention was to expose the subject to two levels ofacute mental stress in a natural situation by performing two fast-paced computer tasks. The subject was instructed about the tasksthat were performed in a randomized sequence. Before the task, thesubject was allowed to practice. Each task lasted 20 min and wasperformed under silent conditions; the first took place at about11.00 h and the second around noon, with a glass of water and a 30-

Age and Physiological Stress Reactions Gerontology 2004;50:383–392 385

Table 1. Description and comparison of the two groups of subjects (mean B SD)

Aged women(63B4 years;n = 13)

Young women(24B3 years;n = 13)

Computer work experience, years 13B1* 7B3Height, cm 164B5* 169B4Body mass index, kg/m² 24.5B2.6* 20.7B1.2Unstimulated, baseline salivary flow rate, ml/min 0.27B0.19 0.48B0.39Maximum unilateral bite force in the molar region, N 247B73* 432B76Maximum EMG activity during clenching, ÌV

Musculus temporalis anteriorMusculus masseter superior

217B106166B69

291B85167B73

Stroop effect: relative reduction of correct answers from REF test to CWT, % 56B24* 31B20

* p ! 0.05 (two-sided t test or Mann-Whitney U test).

min resting break in between. During the break, the subject wasallowed to relax, read, chat, and visit the ladies’ room. The masticato-ry muscle activity was recorded by electromyography (EMG) duringthe tasks. Immediately before and after each task, i.e., with an inter-val of approximately 30 min, saliva was collected again, and the sub-ject rated her level of mental stress on visual analogue scales (VAS).

Another study in the same aged and young subjects dealing withthe activities in forearm, shoulder, and neck muscles during the com-puter tasks is submitted elsewhere [1].

Computer TasksThe subject sat on an office chair adjusted according to height,

back support, and distance to the table. The table was adjusted tosupport the forearms properly, and the mouse mat was placed onelength of the forearm from the edge of the table. A standard computerscreen (17 in; 1,024 ! 768 pixels) was placed in front of the subjectwith its top aligned with the subject’s eyes and the screen-eye dis-tance adjusted individually. The 13 aged women and 5 of the youngwomen used spectacles for computer use. A custom-made vision testconfirmed that all subjects were able to see and read Times NewRoman font characters at font sizes from 11 down to 5.

The subjects performed two tasks with the computer mouse withdifferent levels of mental demand, but the same physical work: amodified CWT [30] and a reference (REF) test developed for a pre-vious study [19, 20]. None of the subjects had previous experiencewith either of the tasks. The CWT served as a mental stress modelwith a high level of mental demand, challenging the subjects’ abilitiesto ignore written information, i.e., an automated psychological pro-cess, while forcing them to respond to colour information only [10,14, 30]. During the CWT, one of the Danish words for ‘red’, ‘green’,‘blue’, or ‘yellow’ was shown on the screen in random order and atdifferent positions with random time intervals (average interval1.3 s, range 0.6–2.0 s). The letters of the word appeared in a colourdifferent from the colour indicated by the meaning of the word.Placed below the word, four icons (7 ! 11 mm) were labeled with thename of the colours in black and always in the same order. The sub-ject was instructed to report the correct colour of the letters of theword by clicking on the corresponding icon as quickly as possible. An

alarm signal sounded, if the answer was either wrong or if theanswering interval was exceeded. In contrast, the REF test onlyrequired the subject to click on a single icon which appeared on thescreen in the same way at the same positions and with the same timeintervals as the CWT, with no alarm signals given. The Stroop effect,i.e., the effect arising from the incongruent challenge of the wordinformation underlying the CWT, was determined as percent reduc-tion in correct answers from REF to CWT (% Stroop effect).

Anthropometrics and Bite ForceThe height was assessed in centimetres according to the subject’s

own report, and the body mass index was calculated as the self-reported weight in kilograms divided by the squared height inmetres.

The unilateral bite force (N) was recorded by means of a straingauge transducer (miniature bite force recorder; Kleven, Oslo, Nor-way) [9] placed on the lower first molar. According to our standardprocedure [4], the transducer was covered with polyvinyl chloridetubes for protection, and the force was measured during maximumclenches (2 s duration) as the stored peak values on the digital display(AME, Mikro-Elektronikk, Horten, Norway). The molar bite forcewas assessed as the mean of three successive trials from the right andthe left side.

VAS for Perceived Task-Related Stress and Self-RatedPerformanceThe subjects rated their feeling of stress after each task on a VAS,

i.e., a 100-mm horizontal scale, with the left end point (0 mm) indi-cating ‘no stress during the task’ and the right end point (100 mm)indicating ‘the highest stress imaginable during the task’. In addition,their general level of stress was also assessed prior to the tasks andcompared between age groups. The subjects also rated their feelingsof the task difficulty and performance on two similar VAS, with theleft end points of the scales indicating ‘not difficult at all’ and ‘nounsatisfying performance’, respectively, and the right end pointsindicating ‘very difficult’ and ‘very unsatisfying performance’, re-spectively.


Saliva Collection and Measurement of the CortisolConcentrationsUnstimulated whole saliva was collected by the draining meth-

od [21], with the subject seated in a relaxed hunched-over position:(1) initially, i.e., baseline collection, time 0 min; (2) immediatelyprior to the first task, time 60 min; (3) immediately after the firsttask, time 90 min; (4) immediately prior to the second task, time 120min, and (5) immediately after the second task, time 150 min. Eachcollection lasted 5 min during which saliva was drained into pre-weighed plastic cups. Each of the five collections was further subdi-vided into two separate periods lasting 2.5 min with separate cups foreach period, resulting in a total of ten saliva-containing cups fromevery subject. Prior to all collections, the subjects were asked to swal-low, and at the end of each 2.5-min collection period to spit theremaining saliva volume into the cup. After weighing the saliva-filledcups (two-digit precision balance), the collected saliva was trans-ferred by a pipette to a vial and cooled to 0°C and stored in a –80°Cfreezer within !2 h.

The salivary cortisol concentration was determined by means ofan immunoassay kit (Salimetrics; Biotech-IgG, Copenhagen, Den-mark). Briefly, saliva samples and standards (0.2–49.7 nmol/l) wereadded to an ELISA plate with anti-cortisol-coated wells. Then horse-radish peroxidase-labeled cortisol was added to all wells, and a reac-tion time of 55 min was allowed. After washing the plate, tetrameth-ylbenzidine was added for 5 min and the reaction terminated by sul-phuric acid. The absorbance, which was inversely related to the con-centration of cortisol in samples and standards, was determined at450 nm. For quality check, a high and a low cortisol standard sup-plied with the kit were processed with all measurements. All sampleswere measured at least twice, and standard curves exceeded the sam-ple concentrations at all times.

The data from the cortisol and saliva measurements were ana-lyzed as follows: reactive salivary cortisol concentration (nmol/l) dur-ing the first 2.5 min after the computer tasks, changes of the level ofabsolute cortisol concentrations (cortisol ¢; nmol/l) from the last2.5 min before to the first 2.5 min after the tasks and from the last2.5 min before to the first 2.5 min after the rest, relative change ofsalivary cortisol concentration from the last 2.5 min before to thefirst 2.5 min after the tasks (cortisol ¢%; nmol/l), unstimulated base-line salivary flow rate before the tasks (mean 2 ! 5 min before eachtask; see table 1), salivary flow rate (ml/min) during the first 5 minafter the computer tasks, and relative change of the salivary flow ratefrom the last 5 min before to the first 5 min after the tasks (saliva ¢%;ml/min).

EMG MeasurementsThe EMG activity from right and left anterior temporalis muscles

and superficial part of the masseter muscles was recorded by meansof bipolar surface electrodes (tin plates, 2 ! 3 ! 10 mm, 10 mmapart; Nicolet Biomedical, Madison, Wisc., USA). The raw EMG sig-nals were amplified with battery-powered differential amplifier mod-ules and filtered with lower and higher cut-off frequencies set at 10and 450 Hz, respectively, and sampled at 1,024 Hz. The amplifiedsignals were recorded on a notebook PC using a DT-9800 USB dataacquisition module and Scope software (Data Translation, Marlboro,Mass., USA). During the 20-min CWT and REF tasks, the EMG sig-nals were recorded for three periods of 5 min. The recordings startedafter 2 min, and three 5-min recordings were subsequently sampled,with 1-min intervals. The root mean square (RMS) trace for each ofthe 5-min EMG recordings was then normalized to the maximum

RMS EMG values obtained during isometric maximum voluntarycontractions of the muscles during maximum clenching (EMGmax)and averaged from each task (mean % EMGmax). The EMGmax wascalculated using a 1-second averaging window. The percentage oftime with EMG gaps (% time), defined as periods with an activity!0.5% EMGmax for at least 200 ms [34], was calculated for each taskfrom the normalized RMS traces. In addition, the averaged normal-ized RMS traces of the temporalis and masseter muscles 500 msbefore, concomitant with, and after the mouse clicks were calculatedfor each task (peak % EMGmax). In this case 20-ms time intervalswere used to calculate the RMS EMG values [1].

StatisticsThe data were analyzed by using conventional statistical methods

(Statistica version 5.0; StatSoft, Tulsa, Okla., USA). The level of sig-nificance was set at 5%. All results are presented as mean values BSD. Kolmogorov-Smirnov analysis was used to test for deviationsfrom the normal distribution. Anthropometric measurements, biteforce, EMGmax, computer work experience, and Stroop effect wereanalyzed by t tests and Mann-Whitney U test for simple differencesbetween young and aged women. In addition, differences in salivarymeasurements and EMG recordings between tasks were analyzed byt tests and Wilcoxon matched-pairs test. The distribution of correctanswers in the tasks between the two age groups was analyzed as a2 ! 2 table by the ¯2 test. For comparisons of perceived mental andphysiological differences between age groups and tasks, Anova (be-tween-group factor, age group, and repeated-measures factor) wasused, including the Tukey honest significant difference test for posthoc comparisons. As the data concerning the mean and peak muscleactivities deviated from the normal distribution, they were logarith-mically transformed prior to analysis (Anova). The relationshipsbetween perceived mental and physiological effects of the tasks andthe Stroop effect were analyzed by means of the Spearman rank cor-relation (rs).

Results

Descriptive Data of the Subjects in the Two Age GroupsThe unstimulated saliva flow rate and the maximum

EMG activity of the anterior temporalis and the superfi-cial masseter muscles did not differ significantly betweenage groups, but the aged women had significantly longercomputer experience, a lower height, a higher body massindex, and a lower maximum bite force (table 1). The dif-ferences in bite forces were most likely not due to agealone [3, 38], but probably also reflected submaximal con-tractions in the older women who in many cases expressedfear of breaking large fillings or damaging bridge construc-tions when biting hard on the transducer between themolar teeth. This fear was not present in the young wom-en or when they were biting in the intercuspal positionduring the recording of the EMGmax.


Table 2. Spearman rank order correlations (rs) for all subjects (n = 26) between perceived mental and physiological effects of tasks with thecomputer mouse

Perceived task-related stress and self-rated difficulty and performance(VAS 0–100 mm)

mental stress

CWT REF task

task difficulty

CWT REF task

unsatisfying performance

CWT REF task

Relative reduction of correct answers from REF task to CWT(% Stroop effect) 0.08 – 0.19 – 0.53* –

Measurements of cortisol and salivaRelative change of salivary cortisol concentration from the level during

the last 2.5 min before to the first 2.5 min after the task (¢%; nmol/l) 0.23 –0.17 0.11 0.21 0.14 0.45*Relative change of salivary flow rate from the level during the

last 5 min before to the first 5 min after the task (¢%; ml/min) 0.36 –0.05 0.37 0.40* 0.24 0.31

EMG activities (RMS EMG)Mean temporalis muscle activity during tasks (% EMGmax) 0.30 0.33 0.21 0.36 0.46* 0.47*Mean masseter muscle activity during tasks (% EMGmax) 0.09 0.06 0.16 0.40* –0.02 0.17Peak temporalis muscle activity at mouse clicking (% EMGmax) 0.22 0.21 0.16 0.28 0.31 0.41*Peak masseter muscle activity at mouse clicking (% EMGmax) 0.26 0.22 0.21 0.54* 0.08 0.26Gaps in temporalis muscle activity during tasks (% time) –0.31 –0.29 –0.20 –0.24 –0.43* –0.56*Gaps in masseter muscle activity during tasks (% time) –0.14 –0.06 –0.26 –0.28 –0.01 –0.02

* p ! 0.05.

Perceived Stress and Task PerformanceThe subjects’ own feeling of mental stress after the

tasks showed no significant correlations with the otherparameters (table 2). However, the subjects’ own rating oftheir task performance was significantly and positivelyrelated to the changes in the salivary cortisol concentra-tions after the REF task and in the temporalis muscleEMG activities (table 2). In addition, the perceived taskdifficulty was significantly and positively associated withthe relative changes of the salivary flow rate as well as themean and peak masseter muscle activities during the REFtask (table 2).

No significant differences between age groups werepresent in the general level of stress rated in the VASbefore the tasks (6 B 7 mm in the aged and 5 B 4 mm inthe younger women). The self-rated stress levels were sig-nificantly increased after the tasks (p ! 0.01, paired t test).However, the aged women felt being more stressed andthe tasks more difficult (table 3). They also rated their per-formance in the tasks as poorer than the young women. Inaddition, the perceived task-related stress and the generalsubjective assessment of the task difficulty were signifi-cantly lower and the subjective satisfaction with the per-formance significantly higher in the REF task than in theCWT (table 3). Moreover, a significantly different distri-bution of correct answers (p ! 0.001) was observed be-

tween the two age groups: in total, the aged women had4,402 correct answers in the REF task and 2,004 correctanswers in the CWT, while the young women had 5,584correct answers in the REF task and 3,859 in the CWT,revealing a significantly higher Stroop effect in the agedwomen (table 1). The correlation analysis revealed a sig-nificant positive coefficient between the relative Stroopeffect and the subjects’ own rating of their task perfor-mance in the CWT (table 2).

Cortisol Concentration and Saliva Flow RateThe normal circadian decline in the cortisol concentra-

tion level was attenuated and modulated in associationwith the tasks (fig. 1). The cortisol response to the stressfultasks was most pronounced in the aged women. Anova onchanges of absolute cortisol concentrations (¢ cortisol) inthe 30-min intervals during or between the tasks showedsignificant (p ! 0.05) differences between age groups andtasks (post hoc comparison p = 0.01 between CWT andrest between tasks; fig. 2): aged women in the CWT 1.17B 2.58 nmol/l, during rest between tasks –0.49 B 1.42nmol/l; young women in the CWT –0.51 B 2.35 nmol/l,during rest between tasks –1.43 B 1.87 nmol/l. No signifi-cant differences were observed between the relativechanges in the cortisol concentrations (¢% cortisol) in theREF test and in the CWT, but there was a significant dif-


Fig. 1. Time course of morning salivary cortisol concentrations inaged and young women in response to acute mental stress. The firstsaliva collection (0 min) was taken at 10.00 h and then immediatelybefore and after two randomized computer tasks (REF and CWT)with different levels of mental stress. The arrows indicate the taskperiods.

Fig. 2. Changes in absolute salivary cortisol concentrations in agedand young women within the 30-min intervals during or between twocomputer tasks. Significant (Anova and post hoc comparison: p !0.05) differences between the concentrations in aged and young sub-jects and in CWT and REST values.

Table 3. Mental and physiological effects of computer tasks (CWT and REF test) in aged and young women (mean B SD)

MBSD Aged women(60–70 years; n = 13)

CWT REF task

Young women(20–30 years; n = 13)

CWT REF task

p a

age groups tasks

Perceived task-related stress and self-rated difficulty and performance, VAS values (0–100 mm)Mental stress 58B22 16B15 38B25 11B8 0.035 0.0002Task difficulty 76B17 28B18 67B25 14B10 0.049 0.0000001Unsatisfying performance 76B16 44B17 55B20 26B18 0.002 0.0002

Measurements of cortisol and salivaSalivary cortisol concentration during the first 2.5 min after the task, nmol/l 5.22B4.16 6.23B5.90 8.77B4.62 7.91B4.06 NS NSRelative change of the salivary cortisol concentration from the last 2.5 min

before to the first 2.5 min after the task, ¢% (nmol/l) 24B59 20B31 –5B26 –15B41 0.031 NSSalivary flow rate during the first 5 min after the task, ml/min 0.40B0.24 0.38B0.23 0.55B0.48 0.48B0.44 NS NSRelative change of the salivary flow rate from the last 5 min before to the

first 5 min after the task, ¢% (ml/min) 61B58 70B62 25B28 –5B24 0.0004 NS

EMG activities (RMS EMG) Mean temporalis muscle activity, % EMGmax 4.4B4.0 3.3B2.3 0.6B0.3 0.8B0.7 0.00001b NSb

Mean masseter muscle activity, % EMGmax 3.0B2.8 2.3B2.3 1.1B1.1 0.8B0.6 0.05b NSb

Peak temporalis muscle activity at mouse clicking, % EMGmax 7.0B8.6 4.1B3.0 1.3B0.9 2.7B4.9 0.001b NSb

Peak masseter muscle activity at mouse clicking, % EMGmax 4.5B4.3 3.4B2.5 2.3B2.4 1.8B1.5 0.019b NSb

Gaps in temporalis muscle activity, % time 24B25 21B15 65B22 59B27 0.00004 NSGaps in masseter muscle activity, % time 39B36 50B29 62B28 70B19 0.049 0.022

a Significance by Anova with Tukey post hoc analysis; the superscript b indicates values after logarithmic transformation.

ference between age groups (table 3). In the aged women,the change in the salivary cortisol concentration was sig-nificantly and negatively correlated with the relativechange in the salivary flow rate after the CWT and posi-tively with the relative change in the salivary flow rate

after the REF test (table 4). In addition, the relativechange of the salivary cortisol concentration was associat-ed with the mean temporalis muscle activity during theCWT in the group of young subjects (table 4).


Table 4. Spearman rank order correlations (rs) between physiological effects of tasks with the computer mouse in 13aged and 13 young women

Cortisol (¢%, nmol/l; relativechange of salivary cortisolconcentration after tasks)

CWT REF task

Saliva (¢%, ml/min; relativechange of salivary flow rateafter tasks)

CWT REF task

Mean temporalis muscleactivity during tasks(% EMGmax)

CWT REF task

Saliva (¢%, ml/min; relative change of salivary flow rate after tasks)CWT

Aged women –0.54* – – – – –Young women 0.32 – – – – –

REF taskAged women – 0.79* – – – –Young women – –0.01 – – – –

Mean temporalis muscle activity during tasks (% EMGmax)CWT

Aged women 0.17 – –0.29 – – –Young women 0.17 – 0.58* – – –

REF taskAged women – 0.15 – –0.12 – –Young women – 0.20 – –0.42 – –

Mean masseter muscle activity during tasks (% EMGmax)CWT

Aged women 0.32 – –0.01 – 0.62* –Young women –0.49 – –0.15 – 0.19 –

REF taskAged women – 0.37 – 0.51 – 0.53Young women – 0.15 – 0.15 – 0.09

* p ! 0.05.

The salivary flow rate increased significantly after theCWT (paired t test: p ! 0.001) and significantly more inthe aged than in the young women (table 3). Moreover,the relative change in the salivary flow rate during thetasks (¢% saliva) was positively related to the perceivedtask difficulty in the REF task (table 2) as well as to thelevel of the mean temporalis muscle activity in the CWT(table 4).

Jaw Elevator Muscle ActivityThe EMG activities (RMS EMG) during the tasks var-

ied systematically between age groups, with higher levelsand shorter relative time with gaps in the aged women,indicating a higher state of activation (table 3). This trendwas most pronounced in the temporalis muscle, whereasthe activities only differed significantly between tasks inthe masseter muscle with higher levels and shorter rela-tive time with gaps during the CWT (table 3). For bothtasks and both jaw elevator muscles, the peak activity atmouse clicking was significantly elevated above the meanlevel as a form of co-activation (Wilcoxon matched-pairstest: p ! 0.001; fig. 3), but the peak activity was signifi-

cantly higher in the aged women (table 3). Also the meanlevels of activity in the temporalis and masseter musclesin the aged women were significantly correlated in theCWT (table 4).

Discussion

The results showed marked differences in the re-sponses to the mental demands from the computer tasksbetween the aged and young women, even if the aged hadsignificantly longer experience with computer work. Thestress-induced relative cortisol response was significantlymore pronounced in the aged, as previously reported [12].The average increase of the salivary cortisol concentrationin response to the tasks was small but significant (0.12 B6.71 nmol/l) and lower than typically observed in labora-tory studies using more severe stressors or as reactions tonatural daily stressors [29]. However, as described byVedhara et al. [33], the mental stress modified the non-linear relation between time of the day and cortisol con-centration, but, in contrast to the prevailing opinion [27],


Fig. 3. Peak average masseter activity in aged and young women inrelation to computer mouse clicks (arrows) during two computertasks: CWT (a) and REF test (b).

the baseline differed with age with lower concentrationsin the older women (fig. 1). It is not likely that the lowerbaseline cortisol concentration could be caused by differ-ences of the anticipated stressors between aged and youngwomen, as the general stress level on the VAS before thetasks did not differ between the age groups. However, theobserved differences in baseline cortisol concentrationscould be an effect of the decreased circadian variation ofthe cortisol secretion in the aged. Another explanationcould be a slight time shift in the circadian variation of thecortisol concentration [8] between age groups. The diur-nal circle is tied to the sleep-wake cycle; a generally earliersleep onset in the aged women would, therefore, producean age-related phase advance of the cortisol circadianrhythm [28]. Moreover, most of the young women lived

closer to the laboratory/university which means that theydid not have to get up as early as the aged women.

As expected from earlier reports, the performance ofthe aged women was less accurate in the two computertasks [6, 19, 23]. It has previously been shown that elderlyhave difficulties in inhibiting word information uponincongruent challenges, as in the CWT [37], and this rela-tion may also account for the age-related increase in theStroop effect observed in this study. Thus, in the presentstudy, the cognitive performance and executive function-ing were reduced in the aged as compared with the youngwomen. In accordance with their actual performance, i.e.,number of correct answers, the aged women generallyassessed their performance as poorer, and they felt morestressed than the younger women did.

Even though the jaw elevator muscles are not directlyinvolved in the moving and clicking of the computermouse, the mean temporalis muscle activity (% EMGmax)increased, and the percentage time with gaps during thetasks (% time) decreased with the feeling of an unsatisfy-ing performance (table 2). In an examination situation,Ruf et al. [26] found a similar positive relation betweenthe level of temporalis muscle activity and the scores on aso-called Breitkopf Helplessness Questionnaire, includingquestions such as ‘Others would presently cope better’, ‘Idon’t think I shall make it’, and ‘I can’t manage any more’,i.e., statements probably reflecting similar feelings asrated in the present VAS for performance. The muscleactivity also peaked in relation to the clicking which wasmost pronounced in the masseter muscle (fig. 3). Thesechanges were more prevalent in the aged women (table 3).Attention-related muscle activity has been shown in themasseter muscle [36] during a complex two-choice reac-tion time task on a visual display unit with minimal phys-ical activity. Such co-activation and increase of the levelof mean muscle activity could be related to a generalincrease in the central drive to the muscles, as has beensuggested in relation to mentally demanding computerwork [20]. The relative task-related increase in saliva flowrate was higher in the aged women (table 3). This relativeincrease was surprising, as mental stress and unease oftendecrease the salivary flow [5, 24]. Nevertheless, the natureof the emotional state associated with stress may have animpact on the response of the salivary reflex. Thus, it hasbeen shown that intense and passionate emotional statesresult in increased salivary flow rates, whereas tense anduneasy emotional states result in decreased flow rates ascompared with a relaxed and pleasant emotional state[11]. In this context, it is important that none of the stres-sors used in this study constituted a threat or danger to the


subjects, although the stressors may have had differenteffects on the aged as compared with the young subjects.Thus, on the basis of the correlations between the task-related relative changes in saliva flow rates and the sali-vary concentrations (table 4), we suggest that the tasksmay have triggered slightly different emotional states inthe two age groups when trying to solve the tasks: in theyoung either an indifferent state (REF) or an intense andpassionate state (CWT) and in the aged a slightly moretense state (CWT) and an intense and passionate state(REF). Moreover, the masticatory-parotid salivary reflexassociated with stimulation of mechanoreceptors locatedin the periodontal ligaments of the teeth [13] may alsohave influenced the results. As previously mentioned, themuscle activity peaked in relation to the clicking whichwas more prevalent in the aged women (fig. 3). Such anactivity is associated with jaw elevation and probablyslight biting. Tsai et al. [31] showed an increased inci-dence of tooth contact in the intercuspal position togetherwith the same type of attention-related increase of the lev-el of activity in the jaw elevator muscles during stressingmental arithmetic. Furthermore, there has been shown apositive correlation between rectified integrated masseterEMG and parotid salivary flow rate [2, 15].

In conclusion, the study showed that solving demand-ing computer tasks induced mental and physiologicalstress and that the magnitude of mental and physiologicalstress was reflected in salivary cortisol concentration, sali-va flow rate, perceived stress and performance, as well asjaw elevator muscle activity. Moreover, aged women weregenerally shown to have a poorer objective performanceand to become more stressed than younger women, find-ings that were reflected in both mental and physiologicalstress markers. Finally, strong interrelations were foundbetween and within the mental and physiological stressmarkers.

Acknowledgments

We gratefully acknowledge the technical assistance of Mrs. J.Lykkeaa as well as the financial support given by Prof. Birgitte Naun-tofte (Department of Oral Medicine, Clinical Oral Physiology, andOral Pathology and Anatomy, School of Dentistry, University ofCopenhagen, Denmark) to Dr. A. Tuxen. The Medical ResearchCouncil is also greatly acknowledged for financial support to Dr. A.Bardow. In addition, the study was financially supported by the Dan-ish Research Agency.

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