ONLINE APPENDIX: LONG MANUSCRIPT VERSION

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 (meanSD: 35.78.8) 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

polyphenolic flavonoids (1,2). The predominant class of flavonoids in cocoa and chocolate

are flavanols with epicatechin being a main representative (1,2). To explore mechanisms

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 lipid peroxidation increases were lowered in healthy men two (20) and

four (21) hours after dark chocolate (20) or flavanol-rich cocoa drink consumption (21) while

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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, and a

5-min mental arithmetic task in front of an audience (25). The TSST evokes reliable stress

hormone increases (25,26). During recovery from stress, subjects remained seated in a 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, eta2=.108, f=.35, Fig. 1A) and epinephrine (interaction group-

by-stress F(1.7/101.0)=4.34, p=.021, eta2=.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, eta2=.100, f=.33; epinephrine: F(1.8/101.8)=4.06, p=.025,

eta2=.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, eta2=.054, f=.24) and epinephrine (interaction group-by-stress F(1.7/99.2)=3.36,

p=.047, eta2=.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, eta2=.053, f=.24; epinephrine: F(1.8/99.9)=3.62,

p=.036, eta2=.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 flavonoid-

rich 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 to

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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 stress-

protective 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

Dark chocolate

(n=31)

Placebo chocolate

(n=34)

P-value

Age [years] 34.5 ± 1.6 36.8± 1.5 .30

Body mass index [kg/m2] 25.0 ± 0.8 25.2 ± 0.7 .84

Mean arterial blood pressure,

MAP [mmHg]

89.6 ± 1.8 91.3 ± 1.6 .48

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]

16.7 ± 1.1 < 5 <.001

Cortisol before chocolate

[nmol/l]

10.1 ± 1.5 9.9 ± 1.2 .90

ACTH before chocolate

[pg/ml]

6.9 ± 1.7 8.6 ± 3.2 .66

Epinephrine before chocolate

[pg/ml]

26.6 ± 3.6 19.9 ± 2.1 .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

21