Note. The figure shows a significant interaction effect when all other effects are held constant. RMSSD – root mean square of successive differences; SCR – skin conductance response; CS+ – stimulus paired with unconditioned stimulus; CS- – stimulus not paired with unconditioned stimulus; UCS – unconditioned stimulus.

Discussion

In the present study, we investigated the effects of the menstrual cycle phase and vagally mediated heart rate variability (vmHRV) on instructed fear conditioning. We found that the menstrual cycle modulated fear discrimination in both startle magnitudes and the SCR. Specifically, an enhanced CS+ vs. CS- differentiation was seen in startle responses in individuals during the luteal phase compared to individuals in the follicular phase. This enhanced discrimination was driven by larger responses particularly to CS+ stimuli. In skin conductance response (SCR) measurements, we observed reduced fear and safety discrimination during the luteal phase in individuals with low resting vmHRV compared to individuals with high vmHRV during the luteal phase and those in the follicular phase.
Firstly, our results indicate higher fear differentiation in startle magnitude during the luteal phase, specifically showing a higher fear-potentiated startle response to the conditioned stimulus (CS+) but not to the unconditioned stimulus (CS-) and intertrial interval (ITI). This finding replicates previous research by Glover et al. (2013), who similarly observed increased fear discrimination in startle measures during the luteal phase. Importantly, this effect was consistent across all participants, regardless of their resting vmHRV. However, we did not find a significant main effect of the menstrual cycle phase on startle magnitude. This contrasts with the results of Armbruster et al. (2018), who reported higher overall startle magnitudes during the luteal phase compared to the follicular phase.
With regard to skin conductance, we observed that individuals with high vmHRV show the same increased fear differentiation between CS+ and CS- (and UCS) in the luteal phase while low vmHRV individuals showed less fear differentiation. In contrast, during the follicular phase, there were no differences in skin conductance fear responses based on vmHRV. Although this three-way interaction term between condition, cycle phase, and vmHRV was only marginally significant, adding it to the model significantly improved the model fit. This indicates a notable enhancement in the explanation of variance in SCR through this interaction. The two-way interaction between phase and condition was not evident until vmHRV was included in the model, which may be the reason why it was not found in previous studies (Carpenter et al., 2022; Milad et al., 2006).  These results emphasize the importance of considering moderating variables that may interact with the menstrual cycle to gain a better understanding of the underlying physiological and psychological changes.
The heightened fear differentiation observed in startle measures across all individuals and in skin conductance in individuals with high vmHRV could potentially be linked to increased estrogen levels during the luteal phase. While some studies have observed increased fear discrimination in individuals with elevated estradiol levels, as seen in skin conductance (Sartin-Tarm et al., 2020) and activation of the amygdala and hypothalamus (Hwang et al., 2015), other studies have not replicated this effect (White & Graham, 2016). In these studies, however, the interpretation of the relation to menstrual cycle phases is limited due to the absence of cycle phase assessment or its exclusion from the analysis, and the reliance on simple median splits of participants' current estradiol levels for group comparisons. It is important to note that high estradiol levels can be observed both during the second half of the follicular phase and throughout the majority of the luteal phase, with a peak during the mid-phase. Consequently, some of the results may be confounded or diluted by including individuals in different phases or predominantly in the luteal phase. To disentangle the effects of the menstrual cycle phase and estradiol and validate the role of estradiol in the amplified fear discrimination during the luteal phase, subsequent studies incorporating evaluations of both phase and hormonal levels would be essential.
We found a positive association of SCR fear discrimination and vmHRV during the luteal phase. Why this association is evident only during the luteal phase of the cycle remains unclear. It is hypothesized that certain individuals possess a lower neuroendocrine reactive threshold to the fluctuating expression of gonadal steroids across the menstrual cycle, contributing to aversive symptomatology during the luteal phase (Nappi et al., 2022). The connection between reduced vmHRV and diminished fear discrimination in skin conductance measures among select participants could potentially reflect this susceptibility. While Schmalenberger et al. (2019) reported a general slight decrease in vmHRV during this phase in a meta-analysis, evidence suggests that this reduction in vmHRV is specifically associated with premenstrual symptoms. Persons who reported high premenstrual symptoms showed the characteristic drop in vmHRV from the follicular to the luteal phase, whereas individuals who did not experience premenstrual symptoms also did not exhibit these fluctuations (Matsumoto et al., 2007). Commonly reported premenstrual symptoms encompass anxiety, irritability, and heightened stress sensitivity (Allen et al., 1991). These symptoms align with the less distinct sympathetic reactivity observed in individuals with low vmHRV during the luteal phase in our study, as similar affective states to those experienced during PMS, such as clinical anxiety, have previously been associated with reduced fear discrimination (Cooper et al., 2018). Furthermore, decreased vmHRV is well-known to also be associated with anxiety and anxious states (Chalmers et al., 2014).
Brosschot et al. (2018) present a framework for these findings. In their Generalized Unsafety Theory of Stress, they argue that prolonged stress responses, even in the absence of stressors (such as chronic anxiety), result from a perceived lack of safety even in ostensibly secure environments. Similar to the Neurovisceral Integration Model of Fear, the authors propose the existence of a default stress response, which, in healthy individuals, is inhibited in safe environments. VmHRV serves as an indicator of this inhibitory capacity. Evidence supporting this theory can be observed in the negative association between vmHRV and SCR in individuals during the luteal phase, as identified in this study (β = -.18). The lower an individual's vmHRV was during this phase, the stronger their SCR response to CS- cues, which signal safety. It's noteworthy that all participants were explicitly informed, as part of the instructed fear acquisition paradigm, that there would be no aversive stimuli associated with this symbol. The reduction of vmHRV during the luteal phase accompanied by the altered skin conductance responses may thus be indicative of the origination of premenstrual symptoms in a phasic reduction of this inhibition of the default stress response. This assumption, however, would have to be verified in a paradigm that additionally assesses symptomatology.  
In a similar way, our results could also be interpreted within Battaglia and Thayer’s (2022) neurovisceral integration theory of fear, which is rooted in the broader neurovisceral integration theory (Thayer & Lane, 2000). This theory highlights the significance of the interplay between the central and autonomic nervous systems in fear processing. This process occurs via a network of interconnected brain structures that facilitate the regulation of a system known as the central autonomic network. A pivotal idea here is that, in healthy individuals, the PFC adeptly gauges the safety or danger of a situation. Through an inhibitory functional connection with limbic structures like the amygdala, fear responses are then dampened in safe scenarios while becoming more pronounced in hazardous assessments. Subsequently, the autonomic nervous system orchestrates the appropriate response, which could manifest as fear or a lack thereof.
The authors emphasize the importance of vmHRV in this context. It may serve as both a real-time indicator of ongoing responses and an index of the strength of the connectivity between the PFC and amygdala on a trait level (Sakaki et al., 2016). This connection, indicated by resting vmHRV, determines an individual's capacity to regulate behaviour effectively and adequately. Battaglia and Thayer (2022) propose that individuals with low resting vmHRV may experience reduced abilities to sufficiently modulate fear responses (Wendt et al., 2019; Wendt et al., 2015).
Our findings partially corroborate this assumption, revealing a connection between vmHRV and effective fear discrimination during the luteal phase of the menstrual cycle. However, this association is only evident in skin conductance and not in startle measures, which partly contradicts the model proposed by Battaglia and Thayer (2022).

Conclusion

In summary, the menstrual cycle does have an overall effect on instructed fear conditioning, but the effects vary depending on the outcome measure and covariates. While there is a higher differentiation during the luteal phase in startle response, skin conductance shows this differentiation only in individuals with high vmHRV during this phase. Low vmHRV individuals show decreased differentiation in this measure during the luteal phase. These findings may provide valuable insights into the origin of the differential intra- and interindividual experience of premenstrual symptoms. Although in the current study, we did not directly assess whether these fluctuations are directly related to the experience of symptoms, our results clearly suggest that the menstrual cycle phase should be considered in fear conditioning paradigms.
 
Acknowledgements
Berenike Blaser was supported by a PhD scholarship from the University of Potsdam (Potsdam Graduate School) with fundings from the Graduate Fund of the State of Brandenburg (Germany). 
 
Declaration of Generative AI and AI-assisted technologies in the writing process
During the preparation of this work the author(s) used ChatGPT 3.5 in order to increase readability of the text. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

References

Allen, S. S., McBride, C. M., & Pirie, P. L. (1991). The shortened premenstrual assessment form. The Journal of Reproductive Medicine, 36(11), 769–772.
Armbruster, D., Grage, T., Kirschbaum, C., & Strobel, A. (2018). Processing emotions: Effects of menstrual cycle phase and premenstrual symptoms on the startle reflex, facial EMG and heart rate. Behavioural Brain Research, 351, 178–187. https://doi.org/10.1016/j.bbr.2018.05.030
Battaglia, S., & Thayer, J. F. (2022). Functional interplay between central and autonomic nervous systems in human fear conditioning. Trends in Neurosciences, 45(7), 504–506. https://doi.org/10.1016/j.tins.2022.04.003
Beckers, T., Hermans, D., Lange, I., Luyten, L., Scheveneels, S., & Vervliet, B. (2023). Understanding clinical fear and anxiety through the lens of human fear conditioning. Nature Reviews Psychology, 2(4), 233–245. https://doi.org/10.1038/s44159-023-00156-1
Benedek, M., & Kaernbach, C. (2010). Decomposition of skin conductance data by means of nonnegative deconvolution. Psychophysiology, 47(4), 647–658. https://doi.org/10.1111/j.1469-8986.2009.00972.x
Blumenthal, T. D., Cuthbert, B. N., Filion, D. L., Hackley, S., Lipp, O. V., & van Boxtel, A. (2005). Committee report: Guidelines for human startle eyeblink electromyographic studies. Psychophysiology, 42(1), 1–15. https://doi.org/10.1111/j.1469-8986.2005.00271.x
Bradford, D. E., Starr, M. J., Shackman, A. J., & Curtin, J. J. (2015). Empirically based comparisons of the reliability and validity of common quantification approaches for eyeblink startle potentiation in humans. Psychophysiology, 52(12), 1669–1681. https://doi.org/10.1111/psyp.12545
Brosschot, J. F., Verkuil, B., & Thayer, J. F. (2018). Generalized Unsafety Theory of Stress: Unsafe Environments and Conditions, and the Default Stress Response. International Journal of Environmental Research and Public Health, 15(3). https://doi.org/10.3390/ijerph15030464
Carpenter, J. K., Bragdon, L., & Pineles, S. L. (2022). Conditioned physiological reactivity and PTSD symptoms across the menstrual cycle: Anxiety sensitivity as a moderator. Psychological Trauma : Theory, Research, Practice and Policy, 14(3), 453–461. https://doi.org/10.1037/tra0001129
Chalmers, J. A., Quintana, D. S., Abbott, M. J.‑A., & Kemp, A. H. (2014). Anxiety Disorders are Associated with Reduced Heart Rate Variability: A Meta-Analysis. Frontiers in Psychiatry, 5, 80. https://doi.org/10.3389/fpsyt.2014.00080
Chapleau, M. W., & Sabharwal, R. (2011). Methods of assessing vagus nerve activity and reflexes. Heart Failure Reviews, 16(2), 109–127. https://doi.org/10.1007/s10741-010-9174-6
Cooper, S. E., Grillon, C., & Lissek, S. (2018). Impaired discriminative fear conditioning during later training trials differentiates generalized anxiety disorder, but not panic disorder, from healthy control participants. Comprehensive Psychiatry, 85, 84–93. https://doi.org/10.1016/j.comppsych.2018.07.001
Globisch, J., Hamm, A., Schneider, R., & Vaitl, D. (1993). A computer program for scoring reflex eyeblink and electrodermal responses written in PASCAL. Psychophysiology, 30, S30.
Glover, E. M., Mercer, K. B., Norrholm, S. D., Davis, M., Duncan, E., Bradley, B., Ressler, K. J., & Jovanovic, T. (2013). Inhibition of fear is differentially associated with cycling estrogen levels in women. Journal of Psychiatry & Neuroscience : JPN, 38(5), 341–348. https://doi.org/10.1503/jpn.120129
Hwang, M. J., Zsido, R. G., Song, H., Pace-Schott, E. F., Miller, K. K., Lebron-Milad, K., Marin, M.‑F., & Milad, M. R. (2015). Contribution of estradiol levels and hormonal contraceptives to sex differences within the fear network during fear conditioning and extinction. BMC Psychiatry, 15, 295. https://doi.org/10.1186/s12888-015-0673-9
Kuhn, M., Wendt, J., Sjouwerman, R., Büchel, C., Hamm, A., & Lonsdorf, T. B. (2019). The neurofunctional basis of affective startle modulation in humans – evidence from combined facial EMG-fMRI. https://doi.org/10.1101/567032
Lonsdorf, T. B., Haaker, J., Schümann, D., Sommer, T., Bayer, J., Brassen, S., Bunzeck, N., Gamer, M., & Kalisch, R. (2015). Sex differences in conditioned stimulus discrimination during context-dependent fear learning and its retrieval in humans: The role of biological sex, contraceptives and menstrual cycle phases. Journal of Psychiatry & Neuroscience : JPN, 40(6), 368–375. https://doi.org/10.1503/140336
Lonsdorf, T. B., Klingelhöfer-Jens, M., Andreatta, M., Beckers, T., Chalkia, A., Gerlicher, A., Jentsch, V. L., Meir Drexler, S., Mertens, G, Richter, J., Sjouwerman, R., Wendt, J., & Merz, C. J. (2019). Navigating the garden of forking paths for data exclusions in fear conditioning research. ELife, 8. https://doi.org/10.7554/eLife.52465
Lonsdorf, T. B., Menz, M. M., Andreatta, M., Fullana, M. A., Golkar, A., Haaker, J., Heitland, I., Hermann, A., Kuhn, M., Kruse, O., Meir Drexler, S., Meulders, A., Nees, F., Pittig, A., Richter, J., Römer, S., Shiban, Y., Schmitz, A., Straube, B., . . . Merz, C. J. (2017). Don't fear 'fear conditioning': Methodological considerations for the design and analysis of studies on human fear acquisition, extinction, and return of fear. Neuroscience and Biobehavioral Reviews, 77, 247–285. https://doi.org/10.1016/j.neubiorev.2017.02.026
Malik, M. (1996). Heart Rate Variability. Annals of Noninvasive Electrocardiology, 1(2), 151–181. https://doi.org/10.1111/j.1542-474X.1996.tb00275.x
Matsumoto, T., Ushiroyama, T., Kimura, T., Hayashi, T., & Moritani, T. (2007). Altered autonomic nervous system activity as a potential etiological factor of premenstrual syndrome and premenstrual dysphoric disorder. BioPsychoSocial Medicine, 1, 24. https://doi.org/10.1186/1751-0759-1-24
Mertens, G., Boddez, Y., Sevenster, D., Engelhard, I. M., & Houwer, J. de (2018). A review on the effects of verbal instructions in human fear conditioning: Empirical findings, theoretical considerations, and future directions. Biological Psychology, 137, 49–64. https://doi.org/10.1016/j.biopsycho.2018.07.002
Merz, C. J., Kinner, V. L., & Wolf, O. T. (2018). Let's talk about sex … differences in human fear conditioning. Current Opinion in Behavioral Sciences, 23, 7–12. https://doi.org/10.1016/j.cobeha.2018.01.021
Milad, M. R., Goldstein, J. M., Orr, S. P., Wedig, M. M., Klibanski, A., Pitman, R. K., & Rauch, S. L. (2006). Fear conditioning and extinction: Influence of sex and menstrual cycle in healthy humans. Behavioral Neuroscience, 120(6), 1196–1203. https://doi.org/10.1037/0735-7044.120.5.1196
Nappi, R. E., Cucinella, L., Bosoni, D., Righi, A., Battista, F., Molinaro, P., Stincardini, G., Piccinino, M., Rossini, R., & Tiranini, L. (2022). Premenstrual Syndrome and Premenstrual Dysphoric Disorder as Centrally Based Disorders. Endocrines, 3(1), 127–138. https://doi.org/10.3390/endocrines3010012
Sakaki, M., Yoo, H. J., Nga, L., Lee, T.‑H., Thayer, J. F., & Mather, M. (2016). Heart rate variability is associated with amygdala functional connectivity with MPFC across younger and older adults. NeuroImage, 139, 44–52. https://doi.org/10.1016/j.neuroimage.2016.05.076
Sartin-Tarm, A., Ross, M. C., Privatsky, A. A., & Cisler, J. M. (2020). Estradiol Modulates Neural and Behavioral Arousal in Women With Posttraumatic Stress Disorder During a Fear Learning and Extinction Task. Biological Psychiatry. Cognitive Neuroscience and Neuroimaging, 5(12), 1114–1122. https://doi.org/10.1016/j.bpsc.2020.04.012
Schmalenberger, K. M., Eisenlohr-Moul, T. A., Würth, L., Schneider, E., Thayer, J. F., Ditzen, B., & Jarczok, M. N. (2019). A Systematic Review and Meta-Analysis of Within-Person Changes in Cardiac Vagal Activity across the Menstrual Cycle: Implications for Female Health and Future Studies. Journal of Clinical Medicine, 8(11). https://doi.org/10.3390/jcm8111946
Schmalenberger, K. M., Tauseef, H. A., Barone, J. C., Owens, S. A., Lieberman, L., Jarczok, M. N., Girdler, S. S., Kiesner, J., Ditzen, B., & Eisenlohr-Moul, T. A. (2021). How to study the menstrual cycle: Practical tools and recommendations. Psychoneuroendocrinology, 123, 104895. https://doi.org/10.1016/j.psyneuen.2020.104895
Sun, Y., Gooch, H., & Sah, P. (2020). Fear conditioning and the basolateral amygdala. F1000Research, 9. https://doi.org/10.12688/f1000research.21201.1
Thayer, J. F., & Lane, R. D. (2000). A model of neurovisceral integration in emotion regulation and dysregulation. Journal of Affective Disorders, 61(3), 201–216. https://doi.org/10.1016/S0165-0327(00)00338-4
Tschudin, S., Bertea, P. C., & Zemp, E. (2010). Prevalence and predictors of premenstrual syndrome and premenstrual dysphoric disorder in a population-based sample. Archives of Women's Mental Health, 13(6), 485–494. https://doi.org/10.1007/s00737-010-0165-3
van der Molen, G. M., Merckelbach, H., & van den Hout, M. A. (1988). The possible relation of the menstrual cycle to susceptibility to fear acquisition. Journal of Behavior Therapy and Experimental Psychiatry, 19(2), 127–133. https://doi.org/10.1016/0005-7916(88)90026-2
Wendt, J., Hufenbach, M. C., Weymar, M., Hamm, A. O., Weck, F., & Ventura-Bort, C. (2020, December 16). Effect of heart rate variability biofeedback on extinction of conditioned fear. Retrieved from osf.io/xa6j9.
Wendt, J., König, J., Hufenbach, M. C., Koenig, J., Thayer, J. F., & Hamm, A. O. (2019). Vagally mediated heart rate variability and safety learning: Effects of instructions and number of extinction trials. Psychophysiology, 56(10), e13404. https://doi.org/10.1111/psyp.13404
Wendt, J., Kuhn, M., Hamm, A. O., & Lonsdorf, T. B. (2023). Recent advances in studying brain-behavior interactions using functional imaging: The primary startle response pathway and its affective modulation in humans. Psychophysiology, e14364. https://doi.org/10.1111/psyp.14364
Wendt, J., Neubert, J., Koenig, J., Thayer, J. F., & Hamm, A. O. (2015). Resting heart rate variability is associated with inhibition of conditioned fear. Psychophysiology, 52(9), 1161–1166. https://doi.org/10.1111/psyp.12456
White, E. C., & Graham, B. M. (2016). Estradiol levels in women predict skin conductance response but not valence and expectancy ratings in conditioned fear extinction. Neurobiology of Learning and Memory, 134 Pt B, 339–348. https://doi.org/10.1016/j.nlm.2016.08.011