Alarm pheromones are airborne chemical signals, released by an individual into the environment, which transmit warning of danger to conspecifics via olfaction. Using fMRI, we provide the first neurobiological evidence for a human alarm pheromone. Individuals showed activation of the amygdala in response to sweat produced by others during emotional stress, with exercise sweat as a control; behavioral data suggest facilitated evaluation of ambiguous threat

Blaise Frederick

David D. Cox

Denis Rubin

Denis Tolkunov

Helmut H. Strey

Jochen Weber

Lilianne R. Mujica-Parodi

Robert Savoy

Yevgeny Botanov

English

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1BRIEF COMMUNICATION
Word Count (Abstract):  67
Word Count (Total): 1,760
References: 13
Figures: 2
Tables: 0
SUPPLEMENTARY MATERIALS
Expanded Methods: 1
Figures (Expanded Methods): 4
Figure (Whole-Brain Activations): 1
Table (Whole-Brain Activations): 1
Caption (Whole-Brain Activations): 1
Second-Hand Stress: Neurobiological Evidence for a Human 
Alarm Pheromone
LR Mujica-Parodi1,2, Helmut H Strey1, Blaise Frederick3, Robert Savoy4, David Cox5,
Yevgeny Botanov1, Denis Tolkunov1, Denis Rubin1, Jochen Weber6
1 Department of Biomedical Engineering, Stony Brook University School of Medicine
2 Department of Psychiatry, Stony Brook University School of Medicine 
3 Consolidated Department of Psychiatry, Harvard University School of Medicine
4 Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General 
Hospital;   Department of Radiology, Harvard University School of Medicine
5 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology
6 Department of Psychology, Columbia University College of Arts and Sciences
2Alarm pheromones are airborne chemical signals, released by an individual into the 
environment, which transmit warning of danger to conspecifics via olfaction. Using 
fMRI, we provide the first neurobiological evidence for a human alarm pheromone. 
Individuals showed activation of the amygdala in response to sweat produced by 
others during emotional stress, with exercise sweat as a control; behavioral data 
suggest facilitated evaluation of ambiguous threat.
The existence of alarm pheromones is well-established in mammals, with animals 
exposed to odors secreted by acutely stressed conspecifics expressing neurobiological 
and behavioral changes that are indistinguishable from their reactions to predators1.   In
recent years, a significant body of research has explored the role of human reproductive 
pheromones, which appear to exist and exert influence on humans in many of the same 
contexts in which they exist for non-human mammals 2. This strong conservation across 
species is biologically suggestive, and predicts that human alarm pheromones may also 
exist and assume functional importance. 
To date, only five studies worldwide have published reports on human alarm 
pheromones.  Two studies3, 4 found that individuals were able to identify, solely by 
smelling sweat collected on axillary pads, whether the sweat donor had been watching a 
frightening versus benign film.  Using a similar collection paradigm with frightening and 
benign films, another study5 found that subjects, when smelling the stress, but not neutral, 
sweat showed improved accuracy in completing a word-association task.  Two studies 
collected sweat from individuals preparing to take a difficult examination with exercise 
sweat as the control.  In one study, females exposed to the stress odor were less likely to 
judge a face as positive when primed with a positive face6, while in the other, auditory 
stimuli provoked an increased startle response7 when subjects breathed sweat collected 
during the stress condition. 
We set out to determine whether inhaling the sweat of people who were 
emotionally stressed produced, in a group of unrelated individuals, neurobiological 
effects associated with emotional arousal.  The chief excitatory area associated with 
emotion is the amygdala8, which has been reliably activated in human neuroimaging 
studies that induce emotional arousal as well as animal studies using rat alarm 
pheromones9.
To obtain human sweat stimuli, we first collected axillary samples obtained from 
144 individuals participating in a stress condition (first-time tandem skydive) and a 
control condition (running on a treadmill for the same duration of time at the same time 
of day). Sweat donors jumped from 4km (13,000 ft.), with one full minute of free-fall at a 
vertical speed of 193km/hr and four minutes under the parachute. Because the tandem-
master controlled the descent, the skydiving condition produced an emotional but not 
physical stressor for our sweat donors, while the exercise condition produced a physical 
but not emotional stressor.   Significant increases in both subject cortisol-levels
(repeated-measures ANOVA, prepost Stress vs. Exercise:  F=39.87, p=0.000, N=40) 
and state-anxiety (paired t-test:  t=10.02, p=0.000, N=40), confirmed that the paradigm
was successful at inducing emotional stress.  The sweat collection and storage protocols 
3were designed to prevent bacterial growth, which gives otherwise odorless sweat its 
characteristic aversive odor.
Axillary samples, once extracted and pooled for each condition, were then used as 
olfactory stimuli for five experiments.  Two fMRI experiments assessed amygdala 
activation as well as possible gender interactions that could indicate confounds due to 
reproductive pheromones.  Since the amygdala is also known to play a role in general 
olfactory processing, for the next two experiments we used a double-blind forced-choice
discrimination task, as well as Likert scales, to determine whether there were odor 
differences (either intensity, valence, or qualitative) between the test and control samples 
that could confound the neurobiological results.  Finally, we tested the behavioral 
implications of the amygdala activation, to investigate how stress sweat affects threat-
perception using psychometric curves generated by subjects’ responses to morphed 
neutral-to-threatening faces.
Subjects for all experiments were screened for anosmia prior to testing.  For the 
fMRI and behavioral experiments, odor presentation was controlled with synchronized 
nasal inhalation; for the odor discrimination experiments, individuals were asked to sniff 
the sample. To control for potential reproductive pheromone confounds, we included 
only heterosexual subjects. Sweat collection and storage protocols, GC-MS validation of 
the sweat extraction methods, evaluation of trial-specific respiratory parameters 
demonstrating compliance with synchronized breathing protocols, as well as other 
experimental parameters, are described in greater detail in the Online Supplementary 
Materials.
fMRI Experiments:  In the first experiment, we presented sweat from 40 male 
donors to 16 subjects (50% female) while their brains were scanned using fMRI. In a 
second (replication) experiment, using different subjects and scanners, we presented 
sweat from an additional 40 donors (50% female) to a different group of 16 subjects  
(50% female) undergoing fMRI, increasing power by doubling the number of stimulus 
presentations.  Because we hypothesized that putative alarm pheromones would modulate 
activity in brain structures related to fear, our analyses focused on the amygdala; all 
values were corrected for multiple-comparisons using small-volume correction (SVC).  
For both experiments, these revealed significant activation of the left amygdala  (1st Exp:
t=4.80/Z=3.68, p(svc)=0.02 [MNI x, y, z=16, 8, 16], N=16; 2nd Exp: t=6.19/Z=4.30,
p(svc)=0.000, [MNI x, y, z=21, 3, 15], N=16; Figure 1) in response to the stress sweat 
as compared to the exercise sweat.  For both experiments, activity was concentrated most 
strongly in the superficial, or corticoid, amygdala (1st Exp: t=4.80/Z=3.68, p(svc)=0.008,
N=16; 2nd Exp: t=6.19/Z=4.30, p(svc)=0.000, N=16)—a region known to have substantial 
olfactory inputs in primates; homologous structures in other mammals have been 
implicated in pheromonal processing10. Because activation patterns were equivalent for 
same-sex and opposite-sex donor-detector pairs (repeated-measures ANOVA: 1st Exp:
F=1.76, p=0.21, N=16; 2nd Exp:  Donor Sex: F=0.21, p=0.65; Detector Sex: F=1.31,
p=0.27; Donor Sex*Detector Sex:  F=0.004, p=0.952, N=16), our findings suggest that 
reproductive pheromones were unrelated to the effect11.
Odor Perception Experiments: While odor intensity and valence can cause 
differential amygdala responses12, 13, subjects rated both odors, using Likert scales ranging 
4from one (“undetectable”/“pleasant”) to ten (“very strong”/“unpleasant”) as mild (Stress:
μ=2.6, s.d.=2.3, Exercise: μ=2.6, s.d.=2.3; Wilcoxon sign-ranks test: Z=1.11, p=0.28,
N=26) and neutral (Stress: μ=4.5, s.d.=1.1, Exercise: μ =4.8, s.d.=0.8; Wilcoxon sign-
ranks test:  Z=1.56, p=0.12, N=26). To investigate whether the stress and exercise sweat 
odors were qualitatively distinct, we conducted a double-blind forced-choice odor 
discrimination experiment, in which 16 subjects (50% female) identified whether 16 
odor-pairs (50% different), randomly presented, were identical or different; subject 
ratings were not significantly different than chance (one-sample t-test: t=0.64, p=0.53,
N=16).  The data suggest that the test and control odors were indistinguishable, and 
therefore rule out non-specific olfactory processing as a likely explanation for amygdala 
activation in response to the Stress vs. Exercise contrast.
Threat-Perception Experiment:  Since data from our previous experiments 
suggested that the observed amygdala activation reflected emotional rather than olfactory 
processing, we tested whether breathing stress sweat vs. exercise sweat from 64 donors 
(50% female) behaviorally affected perception of ambiguous threat. Psychometric curves
were generated from a forced-choice design in which 14 subjects (36% female) indicated 
via response-button whether briefly-presented (200ms) male faces, morphed between 
neutral and angry expressions, were “more neutral” or “more threatening.”  For each
subject, stress and exercise conditions produced psychometric curves, each composed of 
nine points ranging from neutral (10%) to angry (90%), with each point the average of 14 
face presentations. Threat-levels were presented randomly, with experimental conditions
counter-balanced for order. Values for slope, , were calculated for each curve using 
sigmoidal fitting.  These showed sharpened discrimination (mean 43% increase) between
neutral versus angry faces in response to the stress sweat (Stress: =0.192, s.d.=0.101;
Exercise: =0.134, s.d.=0.066; repeated-measures ANOVA:  F=8.30, p=0.01, N=14,
Figure 2).   No differences between conditions were observed for inflection-points
(F=1.35, p=0.27, N=14), suggesting that the effect was specific to increasing accuracy in 
evaluation of ambiguous threat, rather than attribution of threat to neutral stimuli.
Our findings indicate that there may be a hidden biological component to human 
social dynamics, in which emotional stress is communicated via chemosensory cues. 
Sweat collected during an acute emotional stressor, and subsequently presented to an 
unrelated group of individuals, produced significant brain activation in regions 
responsible for emotional processing without conscious perception of distinct odor; 
behavioral data, our own as well as those from previous studies, suggest the emotional 
processing may be specific to enhancing vigilance and sharpening threat-discrimination.
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Figure 1: Breathing stress-derived sweat modulates amygdala, the primary brain 
region associated with emotional arousal. The activation map (a) reflects the 
STRESSEXERCISE contrast, and was produced using height threshold t = 3.7, p<0.001 
(uncorrected) and extent threshold k = 5 voxels.  Whole-brain random-effects analyses 
revealed that the strongest activation in response to this contrast was the left amygdala 
(see Supplementary Online Materials Figure 3 and Table 1 for whole-brain activations); 
there were no significant de-activations.  The MNI coordinates of the maximally 
activated voxel, located in the left amygdala, are [-21, -3, -15].  Corresponding time-
courses for this voxel (b) are shown for STRESSREST and EXERCISEREST
contrasts.
Figure 2: Psychometric curves generated by a forced-choice assessment of 
ambiguous threat show sharpened discrimination between threat and non-threat
while breathing stress-derived sweat. For each subject, data for each condition 
(STRESS, EXERCISE) were fitted with the sigmoid function, where p0 and p0+p define 
upper and lower asymptotes, A0 is the inflection point, and  defines slope. Significant 
differences between conditions were seen for slope, with individuals under the STRESS 
condition more closely approximating ideal discrimination, shown by the dotted line.
Acknowledgements:  This research was supported by funding from the U.S. Army 
Soldier Systems Center Natick, the Office of Naval Research, and the National Institutes 
of Health. The authors declare no competing financial interests. 
Ra. b.
Figure 1 (Mujica-Parodi)
p pe A A0 1 0+ + − −σ ( )
0A
Figure 2 (Mujica-Parodi)


Second-Hand Stress: Neurobiological Evidence for a Human Alarm Pheromone

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Second-Hand Stress: Neurobiological Evidence for a Human Alarm Pheromone

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