Introduction

Traditionally, psychosocial stress has been investigated using laboratory experiments and real-life stressors. The psychosocial stress used in these experiments is public speaking, as this situation induces responses on the psychological level (e.g., emotional or behavioral responses) as well as on the physiological level (e.g., cardiovascular or hormonal responses) (Carrillo et al. 2001). Lecturing to a large number of students, as is the case in a University setting, involves mental, psychological and physical effort, and social evaluation and represents a real-life psychosocial stress (Houtman and Bakker 1987). The studies that have evaluated teaching stress (Weber et al. 2002; Kudielka and Wüst 2008) have mostly been conducted using subjective questionnaires or interview methods (Brouwers and Tomic 2000; Grayson and Alvarez 2008), and few of them were interested in the stress of lecturing (Houtman and Bakker 1987, 1991).

The physiological response to psychological stressors consists particularly of an activation of the sympathetic nervous system (SNS), and a parasympathetic withdrawal. Direct measurements of salivary adrenaline and noradrenaline seem not to reflect sympatho-adrenal medullar (SAM) activity (Schwab et al. 1992). Salivary alpha-amylase (sAA) concentrations have been suggested as an indirect marker for SAM activity (Granger et al. 2007; Nater et al. 2005) and other studies have shown a marked increase in sAA concentrations in response to stressful tasks or procedures, such as a parachute jump (Chatterton et al. 1997) as well as other types of psychological stress-inductions (e.g., pre-examination) (Bosch et al. 2003). sAA has been suggested to be a surrogate for cardiovascular autonomic system balance correlating well with heart rate variability (HRV) parameters (Bosch et al. 2003; Nater et al. 2006).

Heart rate variability is a non-invasive electrocardiographic (ECG) index of the autonomic control of the heart, which has been extensively studied in anxiety (Miu et al. 2009). The analysis in the frequency domain involves the distribution of oscillations, in at least two frequency bands and the determination of the power in each of these bands. Power in the high frequency (HF) band is considered to reflect vagal modulation of the heart, whereas power in the low frequency (LF) band probably reflects a complex interplay between sympathetic and vagal influences (Eckberg 1997). Decreased HRV, indicating a disturbed autonomic nervous system function, has been associated with mental stress, and is a sign of lack of ability to respond by physiological variability and complexity (Horsten et al. 1999). It seems that trait anxiety is related to HRV. In fact, Fuller (1992) has reported a reduction in heart rate, and an increase in LF and HF powers in low trait anxiety as compared to high trait anxiety participants. Ledowski et al. (2005) have shown that anxiety is related to low levels of HRV parasympathetic components during exposure to stressors. However, the results remain debatable (Dishman et al. 2000; Narita et al. 2007). It has also been shown that men and women do not differ in their physiological responses to acute stress, showing little difference in cortisol reactivity (Kirschbaum et al. 1999), and there are controversial data for sex differences in measures of autonomic responses to acute stress (Kudielka et al. 2004; Kelly et al. 2008).

To capture more easily the effects of work related stress, it may be useful to plan studies at the worksite instead of in the more usual clinical laboratory, where environmental factors may act differently (Lucini et al. 2007). This design imposes technical constraints, suggesting the use of simple, non-invasive methodologies, such as spectral analysis of RR variability or saliva samples. It should however been noted that both these techniques provide only indirect indicators of autonomic activity and SAM activity. The objective of the study was thus to evaluate cardiovascular (HRV) and autonomic nervous system activation among professors immediately before and after delivering a lecture to 200 students. We also examined gender differences in alpha-amylase and HRV, because it seems that gender moderates psychophysiological responses to stress (Carrillo et al. 2001).

Methods

Participants

Fifty-two professors (26 males, 26 females) volunteered to participate in this study. Sample characteristics are presented in Table 1. All study participants were healthy and free of cardiovascular diseases as assessed by their medical histories. The answers to the questionnaire administered prior to the experiment indicated that none of the subjects had a physical or mental illness, were pregnant or taking corticosteroids or oral contraceptives. Furthermore, none of the subjects included in the study smoked or admitted to alcohol abuse or to the use of recreational drugs.

Table 1 Sample characteristics by gender
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The body mass index (BMI) of the participants was calculated as weight in kilograms divided by height in meters.

Participants received the saliva sampling materials along with both verbal and written instructions. Prior to data collection, the purpose of this study was explained thoroughly to each subject and informed consent was obtained from each individual, in compliance with the Declaration of Helsinki.

Procedure

The study was carried out over two periods:

  • On the day of the lecture (teaching day)

  • On a non-working day (control day: Sunday). Subjects were specifically requested not to expose themselves to social situations during this day, in order to measure stress markers in a basal, non-stressed condition.

Subjects were also asked to avoid alcohol and caffeinated beverages for the 12 h preceding each period and to abstain from heavy physical activity the day before the investigation.

Hormonal assay

A written protocol on how to collect the saliva was given to each subject. In addition, they were briefed on the collection method: a 4-min unstimulated whole saliva sample was collected at different times in tubes (Sarstedt, Nümbrecht, Germany) since typically used cotton-based devices artificially reduce free cortisol concentrations (Gröschl and Rauh 2006). Samples were taken 30 min after waking (7:00 a.m.), and at 10:00 a.m., 12:00 p.m., and at 8:00 p.m. on the control day (Sunday).

On the teaching day, saliva samples were also taken 30 min after waking (7:00 a.m.), and just before the lecture (10:00 a.m.), just after the lecture (12:00 p.m.), and finally at 8:00 p.m.

To avoid contamination of saliva with blood, participants were instructed not to brush their teeth before the morning saliva samples. Additionally, eating, and drinking beverages containing alcohol, caffeine, or fruit juices were not allowed for 60 min before sampling. The subjects were told not to undergo excessive physical activity for the 48 h prior to the experiment and to refrain from any sporting activities at all 24 h before the study. Besides these restrictions, participants were free to follow their normal daily routines on the sampling days.

The professors were studied when they were lecturing to 200 students. In all cases, the lecture was only the second of the year, and therefore the professors and the students did not know each other. During each lecture, four questions were asked by the students, and there was always a low background noise in the lecture hall.

Saliva samples were stored at −45°C until biochemical analysis. Tubes were centrifuged for 10 min at 3,000 rpm to obtain clear saliva. Saliva volume was estimated by weighing to the nearest milligram and the saliva density was assumed to be 1.0 g ml−1 (Cole and Eastoe 1988). Saliva flow rate (ml min−1) was determined by dividing the volume of saliva by the collection time. The flow rate of saliva of valid samples should not be <0.1 ml min−1. Under basal conditions, the rate of saliva production is 0.5 ml min−1 (Guyton 1991). sAA was assayed using kits (cortisol EIA kit and alpha-amylase assay kit; Salimetrics Inc., State College, PA, USA, respectively). Intraassay maximal coefficients of variation were 6.7% for alpha-amylase. Interassay maximal coefficients of variation were 5.8% for alpha-amylase. Alpha-amylase activity was expressed as international units per milliliter of saliva. The secretion rate of alpha-amylase was calculated by multiplying the saliva flow rate by the concentration of the measured analyte.

Autonomic nervous system indices

Spectral analysis of RR interval variability was used to obtain non-invasive markers of ANS regulation. The RR intervals were collected beat to beat at each experimental sequence (before and after the lecture) using a Bauman cardiofrequency meter (Fleuriez, Switzerland) with a sampling rate of 1,000 Hz from ECG signal. All the RR intervals were edited by visual inspection, to exclude all the undesirable beats, which accounted for <1% in every subject. After 5 min of supine rest, HRV recordings were performed in the lying position for 10 min just before the beginning of the lecture. The same protocol was used after the lecture. Each recording was performed in a room with a quiet atmosphere at ambient temperature and with dimmed lighting. The mean RR interval, time domain analysis, and Fourier transform indices of heart variability were standardized and calculated as previously described (Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology 1996).

Temporal indices

For each recording, we calculated the mean heart rate [beats per minute (bpm)] and the following indices of HRV; the percentage differences between adjacent normal RR intervals more than 50 ms (PNN50), the standard deviation of all RR intervals (SDNN), the square root of the mean of the sum of the squared differences between adjacent normal RR intervals (RMS-SD). These particular variables issued from the time domain analysis represent the parasympathetic activity (PNN50, RMS-SD) and sympathetic activity (SDNN) (Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology 1996).

Fourier transform analysis

Series of 300s or approximately 256 consecutive RR intervals were extracted 5 min before the lecture, and at the end of the lecture using the fast Fourier transform indices. The 5-min RR interval sequence was divided into three blocks. The linear trend was eliminated for each block by linear regression, and a cosine tapered window in the time domain was applied to the data set. Fast Fourier transformation was applied to each block, and the spectral power was estimated as the ensemble average on the frequency domain. The power spectrum analysis was calculated as recommended by the Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology (1996).

The total power of the spectrum (P TOT) indicates the global autonomic nervous system status; the LF power expressed in absolute (LF: 0.04–0.15 Hz) represents a marker of oscillatory sympathetic modulation of the sinoatrial node, whereas the HF component (HF: 0.15–0.50 Hz) is a marker of vagal oscillatory modulation (Pagani et al. 1986). Additional calculations included the ratio LF/HF, which represents an evaluation of the autonomic nervous system balance (Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology 1996). The LF/HF ratio was also calculated as a single number estimate of mental stress (Kristal-Boneh et al. 1995).

The HF and LF indices were also calculated in normalized units (HFnu and LFnu) as 100 × HF/(P TOT − VLF) and 100 × LF/(P TOT − VLF), respectively. This normalization procedure has been shown to allow comparisons between subjects with large differences in total power or VLF noise (Pagani et al. 1997; Iellamo et al. 1999).

Psychometric assessment

The STAI was used to assess personal anxiety (Spielberger et al. 1970). The test consisted of two separate, self-reported scales for measuring the distinct concepts of state and trait anxiety. The STAI is one of the most commonly used scales to measure anxiety with strong psychometric properties, and the trait anxiety reflects a predisposition to anxiety as determined by the personality pattern (Gotlib and Cane 1989). The state and trait scores of STAI represent anxiety level at the time of measurement and that in daily life, respectively. We used the Y-version of STAI translation in French (Gauthier and Bouchard 1993). After informed consent was obtained, each subject completed the trait section of the STAI on the control day, and the trait anxiety score was calculated. Fifteen minutes before their lecture, the teachers completed the state section of the STAI, and the state section score was calculated. Each scale consists of 20 items. Ten minutes after the lecture, the subjects once again were asked to fill out the anxiety questionnaire.

The Perceived Stress Scale (PSS; Cohen et al. 1983), a 14-item instrument that assesses the degree to which recent situations in one’s life are stressful, was administered before the lecture at the same time as the STAI-State.

Statistical analysis

The statistical program package SPSS 15.0 was used for the analysis and treatment of the data. Results are presented as mean ± standard error of means (SEM). Non-parametric tests were used due to the absence of a Gaussian distribution. We used the non-parametric Mann–Whitney U test to compare data obtained in females and males. Non-parametric analyses (Friedman followed by a Wilcoxon’s signed-rank test to determine significant differences) were used in the statistical computations for biological responses during the resting day/the day of the lecture. Statistical power for the various variables examined ranged from 0.7 to 1. A two-way repeated measures ANOVA was used to compare the control and lecture data at four times [2 (day) × 4 (hour of sample)]. We used post hoc Bonferroni test. To correct for sphericity, Greenhouse–Geisser procedure was applied. The relationships between psychological scores and biological parameters were examined using Spearman correlation coefficients. An alpha-level of 5% was used in all the analyses.

Results

Autonomic nervous system indices

Since there were no significant differences between males and females in cardiovascular activity, we did not distinguish between males and females, and the results were grouped together irrespective of gender.

Temporal indices

The average RMS-SD and pNN50 were significantly lower after the lecture than those reported just before the lecture (P < 0.05, power = 0.95) (Table 2). We noted a non significant increase in the SDNN component (P = 0.3, power = 0.7).

Table 2 Descriptive statistics (mean ± SEM) for measures of heart rate variability
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Frequency indices

The HF and HFnu component of HRV were significantly reduced after the lecture (P < 0.05, power = 0.99). No change in the LH and LHnu component of HRV were found (P = 0.4, power = 0.89. The lecture induced a significant increase in the LH/HF ratio (P < 0.05, power = 0.9).

Salivary parameters

Salivary flow rate changes

Salivary flow rates did not change significantly over time, neither the day of teaching nor during the resting day. No differences between the two conditions were detected.

Values were between 0.53 ± 1.7 and 0.54 ± 0.5 ml min−1.

Salivary alpha-amylase responses

Since there were no significant differences between males and females in sAA concentrations, we did not distinguish between males and females, and the results were grouped together irrespective of gender.

Salivary alpha-amylase presents a pronounced diurnal rhythm in accordance with the results of Nater et al. (2007).

Statistical analysis revealed a significant main effect for day of testing and a main effect for sampling time; Alpha-amylase activity increased after the lecture (Fig. 1).

Fig. 1
figure 1

Salivary alpha-amylase activity. Mean values of salivary alpha-amylase during the resting and the teaching days (U/ml). SEM represented with error bars. Teaching day versus resting day: *P < 0.05

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We also noted a significant interaction effect for day of testing and sampling time with lower Alpha-amylase activity at 10:00 a.m. and 12:00 a.m. during the control day as compared to the day of lecturing.

Psychological measures

There were no significant differences in the STAI and PSS scores between males and females. Therefore, we pooled the data.

The STAI-Trait was 38.5 ± 0.4. The STAI-State evaluated 10 min before the lecture was 40.1 ± 0.3. This STAI-State was significantly higher than the one noted on the control day (35.3 ± 0.1; P < 0.05). The STAI-State evaluated 10 min after the lecture was 36.3 ± 0.6. The scores on the PSS (33.1 ± 1.6) were significantly higher (+63%) than those reported by Cohen and Williamson (1988) (18.8 ± 1.2), so present subjects reported a high perceived stress.

Correlations

Correlations were run in order to analyze the relationship between anxiety and changes in physiological parameters. Analyses revealed that the STAI-Trait score, as well as the STAI-state scores were unrelated to physiology. Also, no correlation was noted between the score on the PSS and the biological markers.

No relationship was found between alpha-amylase activity and HRV indices, except the positive correlation noted at the end of the lecture between LH/HF ratio and alpha-amylase (r = 0.36, P < 0.01).

Discussion

The purpose of this study was to examine the effect of lecturing to 200 students on cardiovascular (HRV) and ANS activation and possible gender differences in response in professors. This paper focused on the hypothesis that the combination of cognitive and emotional stressors, such as when a teacher delivers a lecture to 200 students, would induce mental stress, leading to an increase in cardiovascular reactivity, i.e., decreased HRV. While reports are available on chronic stress in lecturers (Bellingrath et al. 2009), very little work related to acute stress, specifically on lecturers, has been conducted. To our knowledge, our study is the first to examine the evolution of alpha-amylase activity and HRV among professors during the day of a lecture. Moreover, the majority of studies on work stress use questionnaires and self-reports, where stress is often measured indirectly by consideration of stressors, outcomes of stress and job dissatisfaction. Recently, investigations have suggested that individual psychophysiological responses to stressors could represent a target for preventive strategies (Chandola et al. 2006).

The evaluation of our subjects through the STAI-Trait and the PSS showed that they were moderately anxious (Gauthier and Bouchard 1993) but that their basal amount of stress was rather high. Regarding psychological responses, the results show that the lecture was emotionally challenging. State anxiety was significantly higher than the one noted on the control day. Ten minutes after the lecture anxiety scores reached levels comparable to the resting day. Thus, the subjects seem to be able to regulate their emotional excitement.

Regarding physiological processes, the lecture was efficient in eliciting enhanced biomarker responses. In fact, there were responses within the situation, i.e., an increase or decrease of a given physiological parameter during teaching as compared to the initial values. These differences are not accounted for by sex differences or by trait anxiety.

It has been reported that high emotional stress is accompanied by changes in HRV (Dishman et al. 2000; Horsten et al. 1999). Moreover, decreased HRV has been associated with mental stress, and with a lack of ability to respond by physiological variability and complexity (Horsten et al. 1999). If the autonomic activation is long lasting, it may influence several physiological processes, and thereby pose a risk of cardiovascular disorders. HF component decreased significantly and LF/HF ratio and HR increased with task application, as we observed in our study, indicating the tilt of autonomic balance toward sympathetic activation. Although the indirect nature of the spectral derived parameters calls for caution in inferring autonomic regulation (Pagani and Malliani 2000), the simultaneous high activity of sAA activity, together with the reduced HF component and increased LF/HF ratio, point to a major rearrangement of autonomic balance toward sympathetic predominance and vagal withdrawal during the lecture, suggesting a reset of major cardiac regulatory mechanisms. Lucini et al. (2002) also reported that psychological stress, as a preparation for a major university examination, induces an enhanced arterial pressure and autonomic adjustments. The main finding of this study is that HRV indices of parasympathetic nervous activity are sensitive indicators of stress during a lecture, is consistent with a number of other studies using either prolonged (Bernardi et al. 2000) or short-term exposure to psychosocial stress (Hjortskov et al. 2004). This last study also suggested a withdrawal of the vagal modulation of the heart during short-term stress. Delaney and Brodie (2000) indicated that after a short exposure to psychological stressors, parasympathetic withdrawal along with an increased sympathetic activity is responsible for an increase in the LH/HF ratio, which was considerably higher compared to that found in our study. We put forward the hypothesis that the stressors imposed on the subjects are the principal parameter which influence the magnitude of this increase.

Our finding that trait anxiety was unrelated to all HRV indices is consistent with a prior report on 92 healthy men and women (Dishman et al. 2000) but is not in agreement with other previous studies (Watkins et al. 1999; Shinba et al. 2008). The absence of this relationship could be due to the healthy state of the subjects in our present study, whose social activities were well maintained in contrast to those of psychiatric patients (Cohen et al. 1999). Another explanation for the difference in the results is the use of different tests to evaluate anxiety [i.e., STAI or Taylor Manifest Anxiety Scale (TMAS)]. In fact, though both scales measure trait anxiety, they are only moderately correlated.

There has been a lack of clear evidence for sex differences in measures of autonomic responses to acute stress; several studies, (as well as ours) show no differences in physiological reactivity (Sgoifo et al. 2003; Kelly et al. 2006), although age-related sex differences in heart rate responses have been observed in response to laboratory social stressors (Kudielka et al. 2004). Although different responses based upon gender were not observed in the our study on the peripheral physiological measures of stress reactivity, the results do not necessarily exclude the possibility of differences between men and women on more central mechanisms related to stress reactivity (Kelly et al. 2008).

To supplement HRV data with a neuroendocrinological approach, we also analyzed sAA levels. Acinar cells in the salivary glands are richly innervated by both sympathetic and parasympathetic nerve fibers, influencing the release of salivary α-amylase by classic neurotransmitters (Turner and Sugiya 2002). Studies in humans and animals have suggested that the activation of the autonomic nervous system leads to a high activity of sAA (Chatterton et al. 1996; Steerenberg et al. 1997). Furthermore, alpha-amylase levels have been found to be associated with cardiovascular function and are suggested to be a surrogate for cardiovascular autonomic system balance (Granger et al. 2007). Bosch et al. (2003) found a significant negative correlation between the parasympathetic-influenced HRV parameter RMS-SD and alpha-amylase levels during stress induction in adults. Furthermore, a positive correlation between alpha-amylase levels and LF/HF as a surrogate for sympathetic tone has been shown (Nater et al. 2006). The same observation was noted in our study, thereby making this parameter a promising indicator for cardiac autonomic function (Schäffer et al. 2008).

In basal conditions in healthy volunteers, sAA activity shows a diurnal profile with a decrease during the first 60 min after waking and an increase during the rest of the day (Nater et al. 2007). Similar to the results obtained by Nater et al. (2007), our study shows that sAA changes in response to but not in anticipation of the lecture (Fig. 1). Early studies also show that sAA is responsive to various types of situations including socially and cognitively oriented laboratory tasks and physical exercise (Chatterton et al. 1996; Nater et al. 2005, 2006). However, the data are controversial. In fact, Borgeat et al. (1984) noted no effect of stress on sAA activity in a laboratory situation, using mental arithmetics as a stressor. Bosch et al. (1996) reported higher levels of alpha-amylase 30 min before an examination compared with baseline, which has been explained by an anticipation of the stressor beforehand. These discrepancies could be explained by the different experimental designs used (Nater et al. 2005). Van Stegeren et al. (2008) also reported increased alpha-amylase activity during anticipation of stressful experiences. These discrepancies could also be explained by the different experimental designs used. In our study, as in the one of Nater et al. (2007), the participants were monitored on a non-working day whereas in other studies as in the one of Van Stegeren et al. (2008), subjects were tested in a laboratory in the afternoon. Moreover, in this last study, the participants came to the laboratory for a psychological experiment, and knew that they were going to be emotionally challenged. These two differences might contribute to the discrepancy of our data and their findings. The number of measurement points was also limited in a lot of studies, concealing a specific reactive pattern of sAA. In our study, we used five measurement points during the teaching day, which allowed us to show an accurate reaction pattern of alpha-amylase. The lack of observed sex differences in sAA levels is also consistent with previous studies (Nater et al. 2007), however the data in the literature are conflicting (Kivlighan and Granger 2006; Van Stegeren et al. 2008).

Studies conducted in a laboratory have the advantage that the experimental conditions are carefully controlled. However, the limitation is that the results and conclusions cannot necessarily be extrapolated to occupational settings. This is why we chose to investigate the stress induced by a lecture at the worksite. By design, because of local constraints, we did not directly measure sympathetic nerve activity, which requires invasive techniques (Pagani et al. 1997), and we limited our autonomic assessment to spectral analysis of RR variability and alpha-amylase activity. Our study found specific emotional responses and physiological response patterns of different physiological systems to lecturing as compared to a resting condition. In fact, HRV and sAA levels, as indicators of cardiac autonomic activity, were altered by a lecture in front of 200 students, these results suggesting a work related stress in these teachers. We did not find sex differences in responses to this psychosocial stress and no effect of trait anxiety on the physiological parameters studied. Further studies should investigate the relationships between HRV and sAA and it would be interesting to investigate the impact of the personality of the teachers in these parameters.