Alarm substances are airborne chemical signals, released by an individual into the environment, which communicate emotional stress between conspecifics. Here we tested whether humans, like other mammals, are able to detect emotional stress in others by chemosensory cues. Sweat samples collected from individuals undergoing an acute emotional stressor, with exercise as a control, were pooled and presented to a separate group of participants (blind to condition) during four experiments. In an fMRI experiment and its replication, we showed that scanned participants showed amygdala activation in response to samples obtained from donors undergoing an emotional, but not physical, stressor. An odor-discrimination experiment suggested the effect was primarily due to emotional, and not odor, differences between the two stimuli. A fourth experiment investigated behavioral effects, demonstrating that stress samples sharpened emotion-perception of ambiguous facial stimuli. Together, our findings suggest human chemosensory signaling of emotional stress, with neurobiological and behavioral effects.

Axillary samples, once extracted and pooled for each condition, were then used as stimuli for four experiments. Two fMRI experiments assessed amygdala activation as well as possible gender interactions that could indicate confounds due to reproductive chemosignals, which have been shown to be sex-specific [13] . The amygdala is not only associated with emotion, but also plays a key role in olfactory processing [28] . To confirm that test and control conditions differed only with respect to emotion, and not perceivable odor, we used a double-blind forced-choice discrimination task, as well as Likert scales, to verify that participants were unable to detect intensity, valence, or qualitative differences in odor between the stress and exercise sweat. Finally, we tested the behavioral implications of the amygdala activation, to investigate how stress sweat affects threat-perception using psychometric curves generated by participants' responses to morphed neutral-to-threatening faces.

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 4 km (13,000 ft.), with one full minute of free-fall at a vertical speed of 193 km/hr and four minutes under the parachute. Because the tandem-master controlled the descent, the skydiving condition produced a predominantly emotional but not physical stressor for our sweat donors, while the exercise condition produced a predominantly physical but not emotional stressor. Significant increases in both participant cortisol-levels (repeated-measures ANOVA, pre−post 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 were designed to prevent bacterial growth, which gives otherwise odorless sweat its characteristic aversive odor.

To date, six studies worldwide have published reports on human stress signaling via sweat. Two studies [19] , [20] 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, one study [21] found that participants, when smelling the stress, but not neutral, sweat showed improved accuracy in completing a word-association task, while another [22] found that stress sweat caused participants to interpret ambiguous expressions as more fearful. 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 face [23] , while in the other, auditory stimuli provoked an increased startle response [24] when participants breathed sweat collected during the stress condition.

The existence of alarm substances in communicating emotional stress via chemosensory cues is well-established in mammals [1] , with animals exposed to odors secreted by acutely stressed conspecifics expressing neurobiological and behavioral changes consistent with increased arousal and threat-assessment [2] – [4] . In recent years, a significant body of research has explored the role of human chemosensory signals for reproductive function, an area that is controversial [5] but which appears to provide some evidence for influence on humans in some of the same contexts in which they exist for non-human mammals [6] – [18] . This conservation across species is biologically suggestive, and predicts that human chemosensory signals for emotional stress may also exist and assume functional importance.

Since data from our two previous experiments suggested that the observed amygdala activation reflected emotion discrimination rather than odor discrimination, we then tested whether breathing stress sweat vs. exercise sweat from 64 donors (50% female) behaviorally affected perception of subtle emotional cues in the evaluation of ambiguous faces. Psychometric curves [33] were generated from a forced-choice design in which 14 participants (36% female) indicated via a computer mouse whether briefly-presented (200 ms) male faces, morphed between neutral and angry expressions, were “more neutral” or “more threatening.” For each participant, 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, σ, 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: σ = 0.192, s.d. = 0.101; Exercise: σ = 0.134, s.d. = 0.066; repeated-measures ANOVA: F = 8.30, p = 0.01, N = 14, Figure 5b ). No differences between conditions were observed for inflection-points (F = 1.35, p = 0.27, N = 14), suggesting that the effect was specific to reducing perceptual noise and thereby increasing accuracy in the evaluation of ambiguous threat, rather than to the attribution of threat to neutral stimuli.

While the fMRI experiments indicate that participants' amygdala were able to distinguish between the sweat of stressed and non-stressed colleagues, it was important to establish whether this activation might be attributable to odor differences between the two conditions [31] , [32] . As shown in Figure 4 , subjects rated both odors, using Likert scales ranging from zero (“undetectable”/“pleasant”) to ten (“very strong”/“unpleasant”) as equivalently 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 conditions had odors that were qualitatively distinct, we also conducted a double-blind forced-choice odor discrimination experiment, in which 16 participants (50% female) identified whether 16 test and control pairs (50% different), randomly presented, were identical or different; participant ratings were not significantly different than chance (one-sample t-test: t = 0.64, p = 0.53, N = 16). The data suggest that participants were not able to consciously distinguish between test and control odors, and therefore rule out simple odor discrimination as an explanation for amygdala activation in response to the STRESS−EXERCISE contrast.

The unmasked activation map (a) reflects the STRESS−EXERCISE contrast, and was produced using height threshold t = 3.7, p<0.001 (uncorrected) and extent threshold k = 5 voxels. The MNI coordinates of the maximally activated voxel, located in the left amygdala, are [x = −27, y = −6, z = −12] (t = 5.21/Z = 3.88; p (small-volume-corrected) = 0.008). Inspection of the mean response to STRESS-REST and EXERCISE-REST contrasts (b) initially appeared to suggest mean deactivation in response to EXERCISE sweat. However, once we factored in the variance (c), it became clear that the effect for the STRESS-EXERICISE contrast was predominantly due to activation in response to the STRESS condition, rather than to deactivation in response to the EXERCISE condition, as only the former showed statistically significant changes from baseline.

In the original experiment, we presented sweat from 40 male donors to 16 participants (50% female) while their brains were scanned using fMRI. In a replication experiment, using different participants and scanners, we presented sweat from an additional 40 donors (50% female) to a different group of 16 participants (50% female) undergoing fMRI, increasing power by doubling the number of stimulus presentations. Because we hypothesized that perception of emotional stress would modulate activity in a brain area related to emotion, 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 (Original Experiment: t = 4.80/Z = 3.68, p (svc) = 0.02 [MNI x, y, z = −16, −10, −18], N = 16; Replication Experiment: t = 5.21/Z = 3.88, p (svc) = 0.008, [MNI x, y, z = −27, −6, −12], N = 16; Figure 2 ) 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 (Original Experiment: t = 4.80/Z = 3.68, p (svc) = 0.008, N = 16; Replication Experiment: t = 5.21/Z = 3.88, p (svc) = 0.008, N = 16)—a region known to have substantial olfactory inputs in primates; homologous structures in other mammals have been implicated in pheromonal processing [29] . Activation patterns were equivalent for same-sex and opposite-sex donor-detector pairs (repeated-measures ANOVA: Original Experiment: F = 1.76, p = 0.21, N = 16; Replication Experiment: Donor Sex: F = 0.21, p = 0.65, N = 16; Detector Sex: F = 1.31, p = 0.27, N = 16; Donor Sex*Detector Sex: F = 0.004, p = 0.952, N = 16), suggesting that reproductive chemosignals, known to be sex-specific in both animals [30] and humans [13] , were not the likely cause. Whole-brain random-effects analyses for the STRESS-EXERICISE contrast ( Figure 3 , Table 1 ) included the amygdala with no significant de-activations.

Discussion

While it is commonly known that information regarding the emotional stress of others is communicated in humans by visual and auditory cues, our findings suggest that humans—like other mammals—may complement this information with chemosensory cues as well. 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.

Our hypothesis and analyses targeted the amygdala, given its critical role in emotion processing; however, areas associated with vision, motor control, and goal-directed behavior also activated in response to the stress sweat. Previous research has established that emotional stimuli not only activate areas of the brain associated specifically with emotion-perception, but also activate sensory areas associated with perception of concomitantly-presented stimuli [34]; this is thought to reflect the increased salience attached to stimuli perceived within emotional contexts. We therefore suspect that increased activation within the cerebellum, BA7, and BA20 most likely resulted from participants‘ enhanced perception during the stress condition of the visual breathing cues (Figure 1a), which required timing inhalation and exhalation to the motion of expanding and contracting rings throughout the experiment.

Because this was the first neuroimaging study to investigate chemosensory cues to emotional stress, we were careful to rigorously control for a number of potential confounds, both methodological and conceptual. Bacterial contamination of sweat contributes to its strong aversive odor; therefore, we developed sample collection methods that would keep the samples as sterile as possible while still preserving chemical components of interest in apocrine sweat. These were validated using gas chromatography-mass spectroscopy (see Materials and Methods). To ensure that differences observed between the two conditions were not due to differences in participant compliance in following the synchronized cues, we also analyzed trial-specific respiratory parameters for the first experiment (see Materials and Methods) and closely monitored participants’ respiration in real-time during each subsequent experiment. The lack of donor sex-detector sex interactions suggests that the effect is unlikely to be consequent to reproductive pheromones released during either of the two conditions. This is a critical point, since a serious limitation of previous studies using stress sweat was the tendency to use male donors and female detectors, which made it impossible to identify sex-effects or eliminate reproductive pheromones as possible confounds to the effect. Finally, replication of the neurobiological findings across two independent fMRI studies with different donor and detector participants suggests the effect is robust to individual variability.

The mean percentage signal change values (Figure 2b) initially appeared to suggest that, as much as stress sweat increased amygdala activity from baseline, exercise sweat reduced it from baseline; therefore, the effect might have been inflated by our choice of a control condition (although using AIR as a control condition would have been even more problematic since AIR, unlike EXERCISE, would not have controlled for sweat odor). However, statistical analyses that consider the variance (Figure 2c) make clear that it was the STRESS condition, and not the EXERCISE condition, that was primarily responsible for the STRESS-EXERCISE effect. For the original fMRI study, the change for STRESS—REST was statistically significant or trend, whether it was calculated using the maximally-activated voxel (t = 1.88/Z = 1.76, p = 0.04, N = 16), ROI analysis for the superficial amygdala (t = 1.48/Z = 1.41, p = 0.08, N = 16), or ROI analysis for the whole amygdala (t = 1.65/Z = 1.55, p = 0.06, N = 16). However, for the EXERCISE-REST contrast, none of the three was statistically significant (for SVC maximally-activated voxel: t = −1.61/Z = −0.27, p = 0.61, N = 16; for the superficial amygdala ROI: t = −0.52/Z = 0.52, p = 0.30, N = 16; for the left amygdala ROI: t = 0.30/Z = 0.31, p = 0.38, N = 16). Exactly the same pattern held for the replication study. Here, the change for STRESS—REST was even stronger, whether it was calculated it using the maximally-activated voxel (t = 3.69/Z = 3.06, p = 0.001, N = 16), ROI analysis for the superficial amygdala (t = 3.23/Z = 2.75, p = 0.003, N = 16), or ROI analysis for the whole amygdala (t = 2.58/Z = 2.29, p = 0.01, N = 16). However, for the EXERCISE-REST contrast, again none of the three was statistically significant (for SVC maximally-activated voxel: t = −1.43/Z = −0.25, p = 0.59, N = 16; for the superficial amygdala ROI: t = 0.36/Z = 0.35, p = 0.36, N = 16; for the left amygdala ROI: t = 0.67/Z = 0.66, p = 0.26, N = 16). Since both original and replication studies show significant differences for the STRESS-REST contrasts, but not for the EXERCISE-REST contrast, it is clear that results obtained for the STRESS-EXERCISE contrast were not driven by participants' responses to the EXERCISE sweat.

The behavioral effect of the STRESS sweat was to sharpen emotional discrimination, rather than to lower thresholds for attribution of threat. Our findings are in line with more recent conceptualization of the amygdala's role, in which the amygdala appears to be not simply a marker for fear, but rather involved in evaluating stimuli for potential threat and then coordinating appropriate responses via its cortical feedback connections (see, for example, [35]). The latter view is consistent with a wide range of fMRI results: for example, the amygdala is activated during conditioning to pain [36]–[38], anticipation of potential pain ([39] but not to pain itself [40]–[42]; likewise, the amygdala is activated in response to social cues to potential threat, such as the aversive outcomes implied by fearful faces [27] but not to unambiguously threatening stimuli such as the object of phobias [43], [44]. As such, one would expect that a chemosensory cue that facilitates the evaluation and discrimination of threat from non-threat would also activate the amygdala, as well as lowering sensory gating for olfactory, visual, and auditory cues that might further inform risk-assessment.

One potential limitation of our study design was that we morphed between only two facial expressions (fear versus neutral); therefore, our study could not confirm whether the sharpened discrimination that we observed extended to all emotional expressions or was restricted specifically for threat. However, results obtained by a recent study [22] argue against generalization. Asked to distinguish between “happy” and “fearful,” in a design similar to ours using morphed facial expressions, participants rated ambiguous faces as “fearful” more frequently in the context of stress sweat, thereby lowering thresholds for detecting fear in others rather than sharpening discrimination. These results suggest that angry and fearful faces communicate distinct types of information that may interact with chemosensory stress cues in complementary ways. Angry faces represent a direct threat, and therefore detection of an anxious colleague's alarm cues may elicit greater vigilance in evaluating whether stimuli signal potential for danger. In contrast, when asked to identify whether faces are fearful in the context of stress sweat, participants are essentially integrating multi-modal sensory cues in detecting colleagues' anxiety, much as auditory cues such as laughter would bias visual perception of an ambiguous smiling faces towards “joy.” Future research, using a within-subjects design, can more directly test this hypothesis.

Previous protocols have sampled sweat in response to stressors such as horror films and pre-examination anxiety. These stressors obviously have the advantage of being easier to administer, but are quite removed from alarm pheromones' evolutionary purpose; i.e., fear associated with imminent physical danger. We chose to address this limitation by using first-time tandem skydives, which have shown to reliably induce acute fear (approaching near-pathological states and including traumatic psychological symptoms such as dissociation, loss of awareness, and time-distortion[45]–[53]), in an ethically acceptable and scientifically-controlled manner. The endocrine and self-report measures confirm that the protocol reliably provoked profound emotional stress in our sweat donors. However, debriefing of our donors and their tandem-masters post-jump indicated that while fear markedly increased during the ascent, peaking in the minutes leading up to exiting the plane and during freefall (≥16 minutes), feelings of relief and/or thrill sometimes followed once the parachute opened and upon landing (≤4 minutes). Donor sweat pads could not be removed until immediately after landing; therefore, it is theoretically possible that our neurobiological and behavioral results resulted from chemosignals emitted in response to non-affect-specific hyper-arousal or thrill, rather than pure fear. However, it is important to note that while alarm substances are well-established neurobiologically, behaviorally, and chemically in a wide number of species, including mammals [54], and therefore their conservation in humans is a reasonable extension, an equivalent “thrill” pheromone has never been reported for any species. Therefore, we believe it is much more likely that participants excreted an alarm substance during the initial fear portion of the protocol, which was retained in the sample even in the face of later relief.