Dark chocolate intake buffers stress reactivity in humans
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Dark chocolate intake buffers stress reactivity in humans
ABSTRACT
Objectives: We investigated the effect of acute intake of dark chocolate on psychobiological stress reactivity in humans.
Background: Flavonoid-rich dark chocolate consumption protects from cardiovascular mortality but underlying mechanisms are not fully understood.
Methods: Healthy men aged between 20 and 50 years (mean SD: 35.7
8.8) were assigned to a single intake of either 50 g of flavonoid-rich dark chocolate (N=31) or 50 g of identically looking flavonoid-free placebo chocolate (N=34). Two hours after chocolate ingestion, both groups underwent an acute standardized psychosocial stress task combining public speaking and mental arithmetic. We measured the stress hormones cortisol, epinephrine, norepinephrine, and adrenocorticotropic hormone (ACTH) prior to chocolate ingestion, before and several times after stress cessation. Plasma levels of the flavonoid epicatechin were also determined. As a psychological stress measure we assessed cognitive stress appraisal.
Results: The dark chocolate group showed a significantly blunted reactivity of the peripheral adrenal gland hormones cortisol (F=6.6, p=.001) and epinephrine (F=4.1, p=.025) as compared to the placebo group. Blunted reactivity of both these stress hormones related to higher plasma levels of epicatechin (p’s .036). There were no group differences in measures relating to central stress reactivity, i.e. the pituitary stress hormone ACTH, the sympathetic neurotransmitter and stress hormone norepinephrine, and cognitive stress appraisal. Potential confounders were controlled.
Conclusions: Our findings indicate that acute flavonoid-rich dark chocolate intake buffers endocrine stress reactivity on the level of the adrenal gland. This suggests a peripheral stress-protective effect of dark chocolate consumption.
Key words: Dark chocolate, stress reactivity, cortisol, epinephrine, ACTH, norepinephrine, flavonoid, epicatechin
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INTRODUCTION
Prospective epidemiological evidence suggests that consumption of dark chocolate or cocoa substantially lowers cardiovascular mortality due to the contained high levels of
underlying the observed flavonoid protection from adverse cardiovascular outcomes, effects on cardiovascular disease (CVD) risk factors have been investigated. Beneficial effects of dark chocolate or flavonoid consumption particularly on blood pressure, blood lipids, the hemostatic system, and endothelial function have been reported after longer-lasting ( 2
weeks) ingestion (2-7), and in part also after acute intake (2,6).
Psychosocial stress is a CVD risk factor (8,9) supposed to promote CVD by inducing
physiological stress responses. In particular, stress induced hyperactivation of the hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS) have been implicated to increase CVD risk, either by direct effects and/or by inducing adverse
changes in intermediate biological risk factors (9-14). Animal studies suggest that short- and
long-term flavonoid administration may protect from adverse stress effects. In rats, acute flavonoid administration reduced water-restraint stress induced HPA axis activation in terms of circulating adrenocorticotropic hormone (ACTH) and corticosteroid as well as
hypothalamic corticosteroid releasing hormone (CRH) mRNA levels (15). Also, several days
of flavonoid administration reduced adrenal hypertrophy induced by repeated immobilization
stress over several days (16). In mice, acute (17) and chronic (18) flavonoid pre-treatment
reduced acute immobilization-stress induced increases in anxiety behavior and impairments in motor activity. In humans, the HPA axis and SNS stress hormones urinary cortisol and
catecholamines were lowered after two weeks of dark chocolate consumption (19). In reaction
to physical exercise
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reactivity of cortisol and ACTH was unaltered (20). In heart patients, consumption of dark
versus control chocolate improved endothelium-dependent vasodilation of coronary arteries in
reaction to cold pressure test (CPT) two hours later (22). In the hitherto only study assessing
endocrine reactivity to acute mental stress after flavonoid consumption, healthy male participants underwent a moderate mental stress task after 6 weeks of consuming either
flavanol containing tea or flavanol-free placebo tea (23). The active tea group showed a faster
decline of salivary cortisol levels after completion of the stress task while baseline and total cortisol secretion, and SNS stress reactivity in terms of blood pressure and heart rate did not
differ between the groups (23).
The aim of this study was to investigate whether a single administration of dark chocolate buffers endocrine reactivity to acute psychosocial stress in healthy men and whether this effect relates to flavonoid plasma levels. Moreover, we wanted to distinguish whether a potential stress-buffering effect of dark chocolate intake would be limited to stress hormones secreted in the periphery only (by measuring the adrenal gland hormones cortisol and epinephrine) or whether measures suggesting a more central effect (by assessing ACTH, norepinephrine, and cognitive stress appraisal) would also be affected.
METHODS
Participants
The ethics committee of the Canton of Zurich, Switzerland formally approved the study protocol. The study was carried out in accordance with the Declaration of Helsinki principles. All participants provided written informed consent before participating.
We intentionally recruited 68 healthy, medication-free, non-smoking men (20-50 years) who reported to consume dark chocolate never or only occasionally and who did not report frequent consumption of flavonoid-containing food or beverages such as black tea, apples, or red wine. Subjects were recruited by aid of the Swiss Red Cross of the State of
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Zurich, and by advertisement. Interested persons underwent a telephone interview to verify eligibility regarding exclusion and inclusion criteria. Subjects with any self-reported acute or chronic somatic or psychiatric disorder or regular dark chocolate consumption were not eligible for the study. Specific exclusion criteria, as obtained by subjects’ self-report, were: alcohol abuse and illicit drug use, any heart disease, varicosis, and thrombotic diseases; elevated blood sugar and diabetes, elevated cholesterol, hypertension, liver and renal diseases, chronic obstructive pulmonary disease, allergies and atopic diathesis, rheumatic diseases, HIV, cancer, chronic pain, sleep disturbances, thyroid disease, and current infectious diseases. If the personal history was not conclusive, the subjects’ primary care physician was contacted for clarification.
Study design and psychosocial stress procedure
We used a placebo-controlled single-blind between-subjects design. Participants and the stress test panel were blind to the study group assignment of a participant while the study coordinator responsible for the age-matching procedure was not. Eligible participants were assigned to either the experimental dark chocolate group or the placebo control group (see below). They were informed that they would receive dark chocolate with either higher or lower cocoa content. To age-match the two subjects groups a first person was randomly assigned to a study group whereas a second person of similar age (±3 years) was assigned to the respective other study group. Of the total of 68 recruited participants 3 scheduled for the dark chocolate group had to stop participation due to technical problems with initial blood sampling (catheter insertion failure (N=2); and catheter occlusion (N=1)) rendering final study samples of 31 subjects in the dark chocolate group and 34 subjects in the placebo chocolate group.
Participants abstained for 48h prior to the experimental session from strenuous physical activity and from consumption of substances that contain polyphenols including dark
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chocolate, red wine, black tea, or apples. In addition, they abstained from consuming milk and milk-containing products as well as coffee (or substances containing caffeine such as coke or energy drinks) and alcohol starting on the evening before the experimental day. They did not consume food and drinks (other than water) in the 2h before reporting to the lab.
Participants reported to the lab at 9:50 am where they received a standardized breakfast (1 soya roll, 20g margarine, 30g honey, and water) at 10:00 am. A venous catheter was inserted at 10:45 am followed 45 min later by the first saliva and blood sampling with subsequent administration of 50g of dark or placebo chocolate required to be consumed instantly. Based on previous observations on the kinetics of plasma flavonoid increases following oral intake of dark chocolate, subjects underwent the psychosocial stressor 2 h after
chocolate ingestion, when plasma flavonoid levels are expected to peak (22,24). We
presumed that dark chocolate ingestion would induce significantly elevated plasma epicatechin levels during the entire stress experiment since flavonoid plasma levels were
found to be still elevated 6 h following oral administration of dark chocolate (24). We applied
the standard protocol of the widely used Trier Social Stress Test (TSST), which combines a short introduction phase followed by a 3-min preparation phase, a 5-min mock job interview,
quiet room.
As stress hormones secreted from the adrenal gland and thus in the periphery only we measured the HPA axis hormone cortisol (secreted by the adrenal cortex) and the SNS
hormone epinephrine (secreted by the adrenal medulla) (27). As hormones indicating a more
central stress effect we measured the HPA axis hormone ACTH secreted by the anterior pituitary and the SNS hormone norepinephrine that is released both as neurotransmitter from sympathetic nerve endings and to a smaller extent as stress hormone from the adrenal medulla
(27). Saliva samples for cortisol measurements were collected before chocolate consumption,
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immediately before and after the TSST, as well as 10, 20, 30, 45, and 60 min after stress cessation in all 65 participants. Blood samples for catecholamine measures were obtained before chocolate consumption, immediately before and after TSST, as well as 10 min after stress cessation. Blood samples for ACTH measures were obtained before chocolate consumption, immediately before and after the TSST, as well as 10 and 60 min after stress cessation. Blood samples for epicatechin analyses were obtained before chocolate administration, immediately before the TSST, and 120 min thereafter. Due to a temporary catheter occlusion immediately after stress in one subject of the placebo group ACTH and catecholamine analyses were performed in 64 participants. In one subject of the chocolate group epicatechin plasma levels could not be determined due to technical problems. Mean arterial blood pressure (MAP) was calculated using an Omron sphygmomanometer from two blood pressure measurements under resting conditions immediately before catheter insertion and immediately before chocolate administration by the formula (2/3*diastolic blood pressure
(BP))+(1/3*systolic BP). All subjects received 175 Swiss Francs after participation as an incentive.
Dark chocolate and placebo chocolate
Chocolat Frey AG, Buchs AG, Switzerland, produced the chocolates for the purpose of this study. The dark chocolate was based on a recipe for a commercially available dark chocolate (“Noir 72%”, Chocolat Frey) with 72% cocoa content. It was produced with a batch of carefully processed cocoa powder to keep the polyphenol content as high as possible and contained 281kcal (13.5g of sugar and 22.8g of fat) for one serving of 50g. The final epicatechin concentration per 50g serving was 125mg. The placebo chocolate was an initially white flavonoid-free chocolate that was dyed with food colors in order to match the color and appearance of the dark chocolate. Moreover, a nutritional chemist from Chocolat Frey flavored the placebo chocolate in order to best match the specific slightly bitter taste of the
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dark chocolate. One serving of 50g of the flavonoid-free placebo chocolate contained 310.5 kcal (13g of sugar and 27g of fat) and 0mg epicatechin. Chocolate composition data were provided by Chocolat Frey and epicatechin concentrations were measured by high performance liquid chromatography in the laboratory Dr. Matt AG, Schaan, Liechtenstein.
Both chocolate types were produced as regular 100 g bars that were packed identically into the commercially available “Noir 72%” wrappings. A small hidden letter on the backside of each chocolate wrapping indicated whether the contained bar consisted of dark or placebo chocolate. Other than that both chocolate types were optically identical with and without wrapping.
Measurements and Data analysis
Biochemical analyses
Biochemical analyses were performed using saliva and blood samples. Blood samples for measurement of plasma ACTH, epinephrine, norepinephrine, and epicatechin were obtained via an indwelling forearm catheter inserted into the non-dominant arm 45 minutes before start of blood sampling. Blood was drawn into EDTA-coated monovettes
(ethylenediaminetetraacetic acid; Sarstedt, Numbrecht, Germany), and immediately centrifuged for 10 min at 2,000g and 4°C; plasma was stored at –80°C until analysis. Saliva for measurement of salivary cortisol levels was collected using Salivette collection devices
(Sarstedt, Rommelsdorf, Germany), which were stored at -20°C until biochemical analysis.
HPA axis stress hormone levels . Flow cytometric determination of ACTH concentrations were performed by Cytolab in Regensdorf, Switzerland, with a commercially available beads immunoassay (Human Pituitary Bead Panel 1, Millipore, Zug,
Switzerland) on a Guava EasyCyte flow cytometer (Millipore, Zug, Switzerland) with a detection limit of 0.5pg/ml. Cortisol concentrations were determined in the biochemical laboratory at the Institute of Psychology of the University of Zurich, Switzerland, with a
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commercially available competitive chemiluminescence immunoassay with high sensitivity of 0.16 ng/ml (LIA, IBL Hamburg, Germany). Intra- and inter-assay variability was less than 10%.
SNS stress hormone and epicatechin levels . To test whether chocolate consumption resulted in increased flavonoid levels, we measured plasma epicatechin concentration in all participants. Plasma epinephrine and norepinephrine, and epicatechin levels were determined by means of high-pressure liquid chromatography (HPLC) using electrochemical detection in the Laboratory for Stress Monitoring (Göttingen, Germany)
(28,29). While catecholamines were quantified after liquid–liquid extraction (28),
conjugated epicatechin in plasma was hydrolyzed enzymatically using beta glucuronidase
and sulfatase, and subsequently extracted with ethyl acetate based on prior methods (29).
The limit of detection was 10 pg/ml for catecholamines, and 5 ng/ml for epicatechin, respectively. Inter- and intra-assay variances were below 5%. Plasma levels below detection limit were replaced by the respective detection limit divided by 2.
Cognitive stress appraisal
To address anticipatory cognitive appraisal processes for the TSST, we assessed the total stress appraisal resulting from primary (i.e., the judgment about the significance of an event as stressful, positive, controllable, challenging, or irrelevant) and secondary (i.e., the assessment of available coping resources and options when faced with a stressor) appraisal processes. For this purpose we used the 16-item questionnaire for Primary and Secondary
Appraisal (PASA) (30) based on the theoretical constructs proposed by Lazarus and
Folkmann (31). The PASA scale Stress Index combines primary and secondary appraisal
providing an integrated measure of transactional stress perception with higher scores
representing higher anticipatory cognitive stress appraisal (30). Cronbach’s alpha was .85 in
our sample for the Stress Index score indicating good reliability.
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Statistical analyses
Data were analyzed using SPSS (version 19.0) statistical software package (SPSS
Inc., Chicago IL, USA). Using G-Power 3 (32) we a-priori defined an optimal sample size
of n=60 to detect a conservatively expected small to medium effect size of f=.18 in general linear models with repeated measures given two groups and the minimum of 3 repetitions
(for catecholamines) with a power of 0.85. All tests were two-tailed with p≤.05 as the significance level. Results are shown as means±SEM. Data were tested for normal distribution and homogeneity of variance in both groups using Kolmogorov–Smirnov and
Levene's test before statistical procedures were applied. We applied Huynh-Feldt correction for repeated measures. Epinephrine and ACTH levels were log-transformed (lg10) to obtain a normal distribution. We used log-transformed epinephrine and ACTH levels for modeling and testing but present untransformed data in tables and figures for reasons of clarity. Body mass index (BMI) was calculated as the ratio of weight in kilograms to height in square meters. Plasma levels below detection limit were replaced by the respective detection limit divided by 2.
Across the two subject groups univariate ANOVAs were calculated to test for differences in group characteristics, stress hormone levels before chocolate consumption, and epicatechin plasma levels.
To test whether dark chocolate consumption induces changes in stress hormone reactivity to acute psychosocial stress, we calculated general linear models with repeated measures. In the main analyses, we entered group (dark chocolate vs. placebo chocolate) as the independent variable and measures of cortisol (7 repetitions), epinephrine (3 repetitions), norepinephrine (3 repetitions), and ACTH (4 repetitions) as repeated dependent variables while controlling for the pre-chocolate baseline of the respective stress hormone as covariate.
Moreover, we controlled for age, body mass index (BMI), and MAP as additional covariates.
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To test whether epicatechin plasma levels prior to the TSST would predict subsequent physiological stress reactivity we recalculated general linear models of the main analyses but entered as the independent variable of interest epicatechin plasma levels assessed immediately before stress induction instead of group. For further statistical details see Supplemental 2.
RESULTS
Group characteristics
Table 1 depicts characteristics of the study groups. As intended, the groups significantly differed in epicatechin plasma levels before and 120 min after stress with measurable levels in the dark chocolate group. Epicatechin levels before chocolate consumption and epicatechin levels in the placebo chocolate group were below the detection limit of 5 ng / ml. The subject groups did not significantly differ in stress hormone levels before chocolate consumption (p’s>.26), nor did they differ in age, BMI, MAP, or psychological measures (p’s>.30).
Physiological stress reactivity in the dark chocolate and the placebo chocolate group
Across all subjects, the TSST induced significant increases in cortisol, ACTH, epinephrine, and norepinephrine (p’s<.001).
General linear models with repeated measures revealed that the dark chocolate group showed a significantly blunted cortisol (interaction group-by-stress
F(2.5/154.8)=7.47, p<.001, eta 2 =.108, f=.35, Fig. 1A) and epinephrine (interaction groupby-stress F(1.7/101.0)=4.34, p=.021, eta 2 =.066, f=.27, Fig. 1B) reactivity to psychosocial stress as compared to the placebo group. Additional controlling for age, BMI, and MAP did not significantly change these results (interaction group-by-stress cortisol:
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F(2.6/155.6)=6.59, p=.001, eta 2 =.100, f=.33; epinephrine: F(1.8/101.8)=4.06, p=.025, eta 2 =.065, f=.26). There were no group differences in terms of ACTH or norepinephrine stress reactivity either without (norepinephrine: p=.27; ACTH: p=.31) or with
(norepinephrine: p=.23; ACTH: p=.31) adjustments for age, BMI, or MAP.
Associations between epicatechin plasma levels assessed immediately before stress and subsequent physiological stress reactivity
Higher epicatechin plasma levels significantly related to lower stress reactivity of the adrenal gland hormones cortisol (interaction group-by-stress: F(2.4/143.5)=3.46, p=.027, eta 2 =.054, f=.24) and epinephrine (interaction group-by-stress F(1.7/99.2)=3.36, p=.047, eta 2 =.053, f=.24) across both subject groups. Again, additional controlling for age, BMI, and MAP did not significantly change these results (interaction group-by-stress cortisol:
F(2.5/145.3)=3.24, p=.032, eta 2 =.053, f=.24; epinephrine: F(1.8/99.9)=3.62, p=.036, eta 2 =.060, f=.25). There were no associations of epicatechin levels with ACTH (p=.28) and norepinephrine (p=.48) stress reactivity.
DISCUSSION
Our study investigated for the first time whether a single administration of flavonoidrich dark chocolate buffers endocrine reactivity to acute psychosocial stress in healthy men and whether this effect relates to plasma levels of the flavonoid epicatechin. We also tested whether a potential stress-buffering effect of dark chocolate intake would be limited to stress hormones secreted in the periphery only or whether the effect would rather be of central nature. The latter assumption was based on recent cell line based experiments suggesting that
cocoa flavanols are capable of crossing the blood-brain-barrier (33).
We found that dark chocolate intake relates to a significantly lowered stress reactivity of the stress hormones cortisol and epinephrine as compared to placebo intake and that this
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buffering effect was associated with higher plasma levels of the flavonoid epicatechin. In contrast, we could not find ACTH and norepinephrine stress reactivity or cognitive stress appraisal to be affected by dark chocolate intake. Notably, in reaction to acute mental stress cortisol and epinephrine are both secreted from the adrenal gland which is a peripheral organ whereas ACTH is centrally released by the anterior pituitary, following hypothalamic CRH
signaling (27). As the largest proportion of plasma norepinephrine results from its activity as
SNS neurotransmitter we interpret norepinephrine as a more central measure of stress but are well aware of its additional peripheral component as adrenal stress hormone. Given this reasoning our findings suggest that dark chocolate intake buffers stress-induced secretion of stress hormones of both major stress systems (i.e. the SNS and HPA axis) in the adrenal gland and thus in the periphery, but not centrally. In line with this, the non-significant difference between our subject groups in terms of anticipatory cognitive stress appraisal suggests that the observed group differences in adrenal stress hormones do not relate to potential alterations in cognitive stress assessment processes. Interestingly, the centrally released ACTH that remained unaltered by dark chocolate or cocoa flavanols did not result in correspondingly high cortisol levels as observed in the control group. Thus, our data suggest that the lower cortisol stress reactivity observed in the dark chocolate group may not result from a potential central chocolate effect via CRH and subsequent ACTH release but rather seems to be a distinct peripheral phenomenon. Our results therefore do not support an overall flavonoid effect on the HPA axis (i.e. on every level of the axis) as observed in the hitherto only animal study investigating effects of acute flavonoid administration on psychological stress-induced
HPA axis activation (15). A possible explanation underlying this divergence may relate to
species differences between humans and rats. Indeed, one human study testing effects of acute flavanol administration on HPA axis reactivity to physical exercise was similarly unable to
find alteration in ACTH levels (20). In contrast to our study, however, that study failed to
detect flavanol effects on cortisol. We can only speculate whether this difference is due to the
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different stimulation procedures, i.e. strenuous exercise vs. mental stress, or whether the study was underpowered (N=14) to detect existing smaller or medium size effects.
Notably, we did not measure cocoa flavanols other than epicatechin. Given the lower effect sizes in the epicatechin calculations compared to the group comparisons, we assume
that other flavanols such as e.g. catechin or procyanidin (2) may add to the stress protective
effects observed in the dark chocolate group. Also, since we did measure epicatechin levels before stress, but not immediately and 10 min after stress, it remains unclear whether associations with epinephrine, or cortisol would have been more pronounced if epicatechin levels had been assessed later.
Despite cell-line based evidence that the main cocoa flavanols catechin and
epicatechin can cross the blood-brain barrier (33), it is still unclear whether catechin and / or
epicatechin can reach the human brain at levels sufficiently high to modify central nervous processes. We speculate that peripheral mechanisms may underlie the observed stress buffering effects of flavonoid-rich dark chocolate intake. Our speculation is based on previous in vitro experiments in human adrenocortical cell lines demonstrating inhibitory effects of various dietary flavonoids on activities of steroidogenic enzymes and, consequently, on the
production and release of cortisol (34-36). Moreover, first findings indicate that dietary
flavonoids might inhibit catecholamine biosynthesis and secretion from the adrenal medulla
(37,38). However, experimental studies are needed to determine exactly whether the specific
mix of cocoa flavanols plays a significant inhibitory role in stress-induced cortisol / epinephrine secretion.
Strengths of our study include the use of a unique placebo chocolate to achieve a best possible match to the experimental dark chocolate and that we used a well-validated
standardized acute psychosocial stress task for inducing the stress response (25,26). We
also enrolled men of a broader age range up to 50 years and age-matched our study groups.
Our study also has limitations. The generalization of our study’s findings might be limited
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to healthy men only; whether our findings also apply to women or cardiovascular patients remains to be elucidated. Moreover, the use of a human sample does not allow us to test for underlying mechanisms of the stress protection observed in the dark chocolate group beyond the measurement of hormones indicating a peripheral vs. a more central flavonoid effect; experimental studies are needed to shed light on mediating mechanisms. Also, it remains unclear whether the observed short-term stress-buffering effects of acute dark chocolate consumption are of clinical relevance in the long run and whether they add to the
cardiovascular protection observed with dark chocolate consumption (1,2,7). In addition,
we can only speculate whether a stress-protective effect of dark chocolate intake also applies to situations of chronic stress exposure and future studies are needed to test for potential dosage effects by varying chocolate serving amounts, or flavonoid concentrations, respectively. Finally, despite promising effects of flavone-containing tea drinking for
several weeks (23) potential stress-protective effects of regular chocolate consumption over
a longer period of time remain to be tested. Clearly, such reasoning would need to consider involuntary weight gain that might ensue the regular consumption of dark chocolate for purposes like improving cardiovascular health.
In sum, our findings indicate that acute flavonoid-rich dark chocolate intake buffers endocrine stress reactivity on the level of the adrenal gland suggesting a peripheral stressprotective effect of dark chocolate consumption. Future cross-sectional and prospective studies are needed to replicate our findings, and to test their clinical relevance, long-term health consequences, as well as generalizability to chronic stress exposure and populations other than healthy men.
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Acknowledgements: There are no conflicts of interest. We thank Dr. Giovanni Balimann and Chocolat Frey for providing the study chocolates and related information; and Leunora
Fejza and Petra Kummer for their help in study conduction.
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Table 1. Medical and psychological group characteristics, stress hormone levels before chocolate administration, and epicatechin plasma levels in the two subject groups
P -value
Age [years]
Dark chocolate
(n=31)
34.5 ± 1.6
Placebo chocolate
(n=34)
36.8± 1.5 .30
Body mass index [kg/m 2 ] 25.0 ± 0.8 25.2 ± 0.7 .84
89.6 ± 1.8 91.3 ± 1.6 .48 Mean arterial blood pressure,
MAP [mmHg]
Stress appraisal [PASA Stress
Index score]
-.79 ± .58 -.45 ± .45 .64
Epicatechin before TSST
[ng/ml]
40.5 ± 2.9 < 5 <.001
Epicatechin 120 min post
TSST [ng/ml]
Cortisol before chocolate
[nmol/l]
ACTH before chocolate
[pg/ml]
Epinephrine before chocolate
[pg/ml]
16.7 ± 1.1
10.1 ± 1.5
6.9 ± 1.7
26.6 ± 3.6
< 5
9.9 ± 1.2
8.6 ± 3.2
19.9 ± 2.1
<.001
.90
.66
.33
Norepinephrine before chocolate [pg/ml]
397.4 ± 25.7 446.1 ± 33.9 .26
Values are given as means ± SEM; SEM: standard error of mean; n: number of subjects
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