Comparing the effects of square, 4-7-8, and 6 breaths-per-minute breathing techniques on heart rate variability, CO₂ levels, and affect.

Josh Marchant, B.S., Mikel Cressman, M.S.

Mentored by: Patrick Steffen, Ph.D. and Inna Khazan, Ph.D.

And a thank you to our wonderful RAs!

Four Breathing Techniques

Evidence for Square/Box Breathing?

• Beşler et al. (2023)
• Sample of 12 soccer players
• Sitting in a zero-gravity chair and doing square breathing increased heart rate variability (HRV)
• Results are inconclusive (lack of a control group and small sample size)
• Moderate effects on mood and cortisol levels during a stressful task, but not on HRV or autonomic functioning (Balban et al., 2023, Bouchard et al., 2012, Röttger et al., 2021)
• Some reduction of post-labor pain (Ahmed et al., 2022)

Evidence for 4-7-8 Breathing?

• Some evidence for helping medical patients with depression and anxiety symptoms (Aktaş & Ílgin, 2023; Pandekar & Thangavelu, 2019)
• Adherence was not measured, lack of clear protocol
• Vierra et al. (2022) found 4-7-8 breathing decreased heart rate variability

Evidence for 6 breaths per minute (5:5 and 4:6) Breathing

• Numerous studies demonstrate increases in HRV, improvements in mood, decreases in blood pressure and decreases in stress (Steffen et al., 2022; Steffen et al., 2021; Edmonds et al., 2009, Grossman et al., 2001; Leyro et al., 2021; Lin et al., 2014; Van Diest et al., 2014)
• Breathing at around 6 breaths per minute is close to resonance frequency for most people (Lehrer et al., 2003)
• Effective physiological regulation

Heart Rate Variability (HRV)

• Beat-to-beat variations in heart rate
• Time Domain: Root Mean Squared Successive Differences (RMSSD)
• Frequency Domain: High frequency (HF) at normal breathing rate, low frequency (LF around 6 breaths per minute )
• Degree to which autonomic nervous system can be modulated to meet environmental demands (Appelhans & Luecken, 2006)
• Marker of parasympathetic functioning (rest and digest)
• Low HRV is implicated in numerous forms of psychopathology (Beauchaine & Thayer, 2015)

End Tidal CO₂ (PETCO₂)

• Measure of carbon dioxide released at the end of an exhalation
• Significant increases signify under-breathing
• Significant decreases signify over-breathing (Gilbert, 2005)

Blood Pressure

• Systolic and diastolic
• Measured by an automated blood pressure cuff on the non-dominant arm

Affect

- Scale of Positive and Negative Experience (SPANE)

- Has demonstrated good reliability, convergent validity, and divergent validity with other measures of mood/affect (Jovanović et al., 2020).

Design

• N = 84 college students (based on power analysis, assuming effect size of 0.17 and power of 0.8)
• Eligible if they had typical cardiovascular functioning and no prior formal breathing experience
• Within-subjects design
• 10-minute baseline, followed by 10 minutes of each breathing technique with 3-minute breaks in between (last 5 minutes analyzed)
• Counterbalanced groups
• A: 5:5, 4:6, Square, 4-7-8
• B: 4:6, Square, 4-7-8, 5:5
• C: Square, 4-7-8, 5:5, 4:6
• D: 4-7-8, 5:5, 4:6, Square

​

Design

• Participants blinded to group
• Instructed by research assistants not to breathe too deeply and to breathe into the diaphragm
• Due to equipment error, final sample for analyses was N = 78 (7% missing)
• Primary analysis with mixed effects autoregressive modeling, treating breathing technique and counterbalanced group as fixed effects with participant as a random effect

​

​

Descriptive statistics for demographic variables.

Percentage adherence by breathing technique.*

*ICCs for counting breathing rate with respiration belt were 0.87 (5:5), 0.75 (4:6), 0.80 (square), and 0.74 (4-7-8).

Hypothesis 1

• H1. Compared to square and 4-7-8 techniques, breathing at 6 bpm will increase RMSSD and LF-HRV. Square and 4-7-8 breathing will increase VLF-HRV by more than 6 bpm.
• H5. There will be no significant HRV or PETCO₂ differences between the two 6 breaths-per-minute conditions (4:6 and 5:5).

*Counterbalanced group was not associated with outcomes

Potentially unhealthy

*VLF is not as accurate during 5-minute measurement segments

Hypothesis 2

• H2. Breathing at 6 breaths per minute will reduce both diastolic and systolic blood pressure more than 4-7-8 or square breathing.

​

• We observed no statistically significant effect of any breathing condition on blood pressure.
• Low baseline: an SBP: 107, Mean DBP: 62
• Prior studies have shown significant reductions in blood pressure during slow breathing over multiple weeks (Grossman et al., 2001).

Hypothesis 3

• H3. 4-7-8 and square breathing will increase CO2 retained in the lungs as measured by end-tidal CO2 (PETCO2), signifying under breathing. We hypothesize that breathing at 6 breaths per minute will slightly reduce PETCO2 levels.

- Machine was not zeroed before some CO2 measurements were taken, leading to some inaccurate baseline results. However, change scores were accurate.

- Excluding participants whose baseline was not in the normal (35-45 range) results in nearly the same Beta and p-values.

Hypothesis 4

• H4. Breathing at 6 breaths per minute will increase affect balance scores on the Scale of Positive and Negative Experience (SPANE) when compared to square or 4-7-8 breathing, indicating increased positive affect.

Conclusions

• 5:5 and 4:6 increased HRV more than square or 4-7-8 breathing during the technique.
• This suggests a strengthening of homeostatic reflexes/physiological regulation.
• Square and 4-7-8 breathing demonstrated some small increases in HRV but were much harder to do. These techniques also increased VLF-HRV compared to 5:5 and 4:6, which could be potentially unhealthy.
• There was no impact on mood in 4:6 and 5:5 breathing, indicating that people may not consciously be aware of the physiological impacts (at least at first). Square and 4-7-8 breathing negatively impacted mood.
• People tended to over-breathe during 5:5 and 4:6 breathing. It is important to monitor CO₂ and help people not over-breathe during 6 bpm breathing.
• Gently work down to 6 breaths per minute

Exploratory – email sent after study completion; only half of the sample responded

Limitations

• Sample size of 78 may not have been large enough to detect smaller HRV effects for square and 4-7-8 breathing.
• Very homogenous sample.
• No measure of the effects of breathing over time (e.g., 4:6 breathing every day for a month); comparing pre to post baseline.
• Some equipment issues with CO2 measures.
• This led to 7% missing data, but our statistical methods were able to handle missing data.

Main Takeaway

• Breathing at 6 breaths per minute is more effective than square or 4-7-8 breathing at regulating the nervous system and increasing physiological well-being.

References

• Ahmed, A., Hassan, S., & Elsaba, H. (2022). Effect of four-square breathing exercise on after pains, initiation of breastfeeding, and satisfaction with intervention among postpartum mothers. Assiut Scientific Nursing Journal, 10(29), 11–22. https://doi.org/10.21608/asnj.2022.120643.1319
• Aktaş, G. K., & İlgin, V. E. (2023). The effect of deep breathing exercise and 4–7-8 breathing techniques applied to patients after bariatric surgery on anxiety and quality of life. Obesity Surgery, 33(3), 920–929. https://doi.org/10.1007/s11695-022-06405-1
• Appelhans, B. M., & Luecken, L. J. (2006). Heart rate variability as an index of regulated emotional responding. Review of General Psychology, 10(3), 229–240. https://doi.org/10.1037/1089-2680.10.3.229
• Balban, M. Y., Neri, E., Kogon, M. M., Weed, L., Nouriani, B., Jo, B., Holl, G., Zeitzer, J. M., Spiegel, D., & Huberman, A. D. (2023). Brief structured respiration practices enhance mood and reduce physiological arousal. Cell Reports Medicine, 4(1), 100895. https://doi.org/10.1016/j.xcrm.2022.100895
• Beauchaine, T. P., & Thayer, J. F. (2015). Heart rate variability as a transdiagnostic biomarker of psychopathology. International Journal of Psychophysiology: Official Journal of the International Organization of Psychophysiology, 98(2 Pt 2), 338–350. https://doi.org/10.1016/j.ijpsycho.2015.08.004
• Beşler, H. K. (2023). The acute effect of zero gravity chair and breathing exercises on soccer players HRV. Performance Analysis in Sport and Exercise, 2(1), Article 1.
• Bouchard, S., Bernier, F., Boivin, É., Morin, B., & Robillard, G. (2012). Using biofeedback while immersed in a stressful videogame increases the effectiveness of stress management skills in soldiers. PLoS ONE, 7(4), e36169. https://doi.org/10.1371/journal.pone.0036169
• Gilbert, C. (2005). Better chemistry through breathing: The story of carbon dioxide and how It can go wrong. 33.
• Grossman, E., Grossman, A., Schein, M., Zimlichman, R., & Gavish, B. (2001). Breathing-control lowers blood pressure. Journal of Human Hypertension, 15(4), 263–269. https://doi.org/10.1038/sj.jhh.1001147
• Lehrer, P. M., Vaschillo, E., Vaschillo, B., Lu, S.-E., Eckberg, D. L., Edelberg, R., Shih, W. J., Lin, Y., Kuusela, T. A., Tahvanainen, K. U. O., & Hamer, R. M. (2003). Heart rate variability biofeedback increases baroreflex gain and peak expiratory flow. Psychosomatic Medicine, 65(5), 796. https://doi.org/10.1097/01.PSY.0000089200.81962.19
• Leyro, T. M., Versella, M. V., Yang, M.-J., Brinkman, H. R., Hoyt, D. L., & Lehrer, P. (2021). Respiratory therapy for the treatment of anxiety: Meta-analytic review and regression. Clinical Psychology Review, 84, 101980. https://doi.org/10.1016/j.cpr.2021.101980

References

• Lin, I. M., Tai, L. Y., & Fan, S. Y. (2014). Breathing at a rate of 5.5breaths per minute with equal inhalation-to-exhalation ratio increases heart rate variability. International Journal of Psychophysiology, 91(3), 206–211. https://doi.org/10.1016/j.ijpsycho.2013.12.006
• Pandekar, P. P., & Thangavelu, P. D. (2019). Effect of 4-7-8 breathing technique on anxiety and depression in moderate chronic obstructive pulmonary disease patients. International Journal of Health Sciences, 5.
• Röttger, S., Theobald, D. A., Abendroth, J., & Jacobsen, T. (2021). The effectiveness of combat tactical breathing as compared with prolonged exhalation. Applied Psychophysiology and Biofeedback, 46(1), 19–28. https://doi.org/10.1007/s10484-020-09485-w
• Steffen, P., Bartlett, D., Channell, R., Parsell, K., Giordano, N., & Branson, K. (2022). How to breathe to improve HRV: Low and slow breathing improves HRV more than deep breathing except when using a pacer [Preprint]. In Review. https://doi.org/10.21203/rs.3.rs-1394127/v1
• Steffen, P. R., Bartlett, D., Channell, R. M., Jackman, K., Cressman, M., Bills, J., & Pescatello, M. (2021). Integrating breathing techniques into psychotherapy to improve HRV: Which approach is best? Frontiers in Psychology, 12, 624254. https://doi.org/10.3389/fpsyg.2021.624254
• Van Diest, I., Verstappen, K., Aubert, A. E., Widjaja, D., Vansteenwegen, D., & Vlemincx, E. (2014). Inhalation/exhalation ratio modulates the effect of slow breathing on heart rate variability and relaxation. Applied Psychophysiology and Biofeedback, 39(3–4), 171–180. https://doi.org/10.1007/s10484-014-9253-x
• Vierra, J., Boonla, O., & Prasertsri, P. (2022). Effects of sleep deprivation and 4-7-8 breathing control on heart rate variability, blood pressure, blood glucose, and endothelial function in healthy young adults. Physiological Reports, 10(13), e15389. https://doi.org/10.14814/phy2.15389