Root mean square of successive differences (RMSSD) is not a valid measure of parasympathetic reactivity during slow deep breathing Author: M Khawar Ali1,2, Lijun Liu1, Amer Hussain1, Difei Zheng1,3, Marzia Alam1, Ji-Hong Chen1,4, Jan D Huizinga1,2,4 Affiliation: <sup>1</sup> Department of Medicine, Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, Ontario, Canada. <sup>2</sup> Department of Engineering, School of Biomedical Engineering, McMaster University, Hamilton, Ontario, Canada. <sup>3</sup> Honours Biology Pharmacology Coop Program, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada. <sup>4</sup> Department of Medicine, Division of Gastroenterology, McMaster University, Hamilton, Ontario, Canada. Conference/Journal: Am J Physiol Regul Integr Comp Physiol Date published: 2023 Jan 30 Other: Special Notes: doi: 10.1152/ajpregu.00272.2022. , Word Count: 261 Deep breathing exercises are the second most used complementary health approach in the United States. Two HRV parameters, the root mean square of successive differences (RMSSD) and the respiratory sinus arrhythmia (RSA), but they are often not in agreement. Our purpose was to determine the cause of disagreement and we conclude that RMSSD cannot be used, although it is used most often. We investigated HRV parameters in 21 subjects during baseline, deep breathing and recovery. Here we show that RMSSD as a measure of parasympathetic reactivity and HRV is unreliable; it does not reflect the known increase in HRV during deep breathing as determined by RSA. We observed a decrease in RMSSD despite a marked increase in HRV as determined by RSA in both healthy subjects and patients with functional bowel disorders. We show that RSA captures all aspects of HRV, whereas successive differences in heart rate intervals are but a small part of HRV with decreasing variability during deep breathing. Using RSA, we present a unique demonstration of the increased heart rate variability during deep breathing that may become an essential tool for researchers and clinicians. Hence, during deep breathing, RMSSD cannot be used to assess parasympathetic reactivity, the use of RSA is recommended. The use of RMSSD in influential studies has led to erroneous conclusions related to parasympathetic reactivity during deep breathing. Its continued use will undervalue the role of the autonomic nervous system in deep breathing, and it will undermine deep breathing as a treatment. Keywords: Autonomic nervous system; RMSSD; RSA; deep breathing; heart rate variability. PMID: 36717167 DOI: 10.1152/ajpregu.00272.2022