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 Table of Contents  
Year : 2018  |  Volume : 2  |  Issue : 4  |  Page : 111-118

Heart rate variability: An overview and a few immediate/short-term assessments

Association Nuytrage, San Benedetto del Tronto, Italy

Date of Web Publication30-Oct-2019

Correspondence Address:
Dr. Cavezzi Attilio
Eurocenter Venalinfa, Viale Dello Port 14, 63074 San Bendetto del Tronto (AP)
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/hm.hm_27_19

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Background: Heart Rate Variability (HRV) is a parameter used to investigate the autonomous neural system (sympathetic and parasympathetic system). Reduced HRV is a risk factor which correlates with physical and psychological stress, psychoneuroendocrineimmunology dysregulation as well as with aging. HRV is considered a reliable parameter to investigate psycho-physical resilience, the latter being considered a key-factor for human longevity and the final target of hormesis pathways as well. Different chemical, physical, psychological stressors may interfere with HRV. Aims: The aim of our observational study was to assess the possible changes in HRV changes induced by a few targeted stimulations: breathing, maqui nutraceutical intake, physical activity and thermal stress, and maqui nutraceutical intake. Patients and Methods: An observational study was performed on 4 groups of subjects, for a total of 112 individuals, who underwent HRV analysis after administration of different stressors/stimuli, such as breathing, thermal stimulation, Maqui 500 ® nutraceutical intake and physical activity. The investigated parameters were: SDNN, RMSSD, MHRR, mean heart rate (MHR) and the three frequency-domain based bands (VLF, LF, HF). Results: Our outcomes demonstrate that HRV assessment is a useful and reliable investigation to highlight and monitor the effects of these stressors. Breathing was the stimulus which induced the most significant variation in HV parameters. Conclusions: Notwithstanding the short duration of the follow-up, immediate/short-term HRV assessment showed statistically significant variations of the main parameters (e.g. SDNN, RMSSD and frequency bands) in a few cases after stimuli exposure. Studies including larger cohorts and longer follow-up are needed and justified to corroborate our outcomes furthermore.

Keywords: Breathing, heart rate variability, hormesis, maqui, physical activity, psychoneuroendocrineimmunology, resilience, sauna

How to cite this article:
Roberto C, Giuseppe DI, Attilio C. Heart rate variability: An overview and a few immediate/short-term assessments. Heart Mind 2018;2:111-8

How to cite this URL:
Roberto C, Giuseppe DI, Attilio C. Heart rate variability: An overview and a few immediate/short-term assessments. Heart Mind [serial online] 2018 [cited 2021 Sep 23];2:111-8. Available from: http://www.heartmindjournal.org/text.asp?2018/2/4/111/270066

  Introduction Top

In recent years, there has been increased scientific research using heart rate variability (HRV) to assess several human activities and psychophysiological interventions which ultimately impact the neurovegetative system. In the PubMed scientific search engine alone, mainly based on the MEDLINE database, the publications containing the term “Heart Rate Variability” have gone from 182 in 1990 to 1620 at the end of 2018,[1] confirming a growing interest around this parameter as an index of autonomous neural system (ANS) function.

Similarly, there has been an increase in the production of HRV biofeedback devices with diagnostic functions and utility for biofeedback training and intervention. Wearable technologies and easily usable devices nowadays allow to obtain data and information to modulate daily activities and more interestingly to improve lifestyle, nutrition, wellness, and health.

The uniqueness and complexity of the human neurovegetative system has led to an effort to interpret daily, physiological and pathological changes in the balance between sympathetic and parasympathetic (vagal) system. HRV proved to be a reliable parameter which assesses the overall variations in the ANS in health and disease. Exposure to different types of stressors reflects on HRV changes, and current devices may monitor the outcomes of inner and/or outer chemical/physical/psychological perturbations on the human organism as a whole. Furthermore, interventions on the subject's nutritional and lifestyle profile, aimed to reestablish the healthy homeodynamics (e.g., physical activity, breathing, calorie restriction, fasting, special nutrients, thermal exposure, and meditation) may be adequately assessed and monitored through HRV.

HRV is the beat-to-beat variation in time intervals between the contractions of the heart,[2] or the distance between systolic phases. HRV is made up of variations in the time intervals between consecutive heart beats which are called interbeat intervals and are measured in milliseconds.[3] Specific algorithms are used to calculate the intervals between the R-R peaks, generating numerical sequences, as well as to derive graphs, indices, and ratios.

Contrary to what was thought in the past, a healthy heart does not beat like a metronome but has complex dynamics in which there are periods of acceleration of the beat, with a decrease in time in the RR interval, and others of deceleration, with an increase in time in the RR interval. Time intervals between heartbeats that have significant variations in their dynamics, during rest periods, show a high efficacy of vagal tone, reflecting the individual's better ability to adapt to physical and psychological challenges (resilience).

A healthy heart shows a high complexity in its oscillations and adapts quickly to sudden physical and psychological changes through the efficacy of its cardiac regulation systems, achieving stability through change. Conversely, a diseased heart shows regularity and low complexity, responding in an ineffective way to sudden changes, due to the breakdown of the regulation mechanisms.[4] More importantly, these heart rhythm variations strictly reflect several overall psychophysical conditions. In fact, reduced HRV is a relevant risk factor which is associated with vulnerability, physical and psychological stress, and illness.[5]

The cardiovascular apparatus integrates different control systems, and its oscillatory models are unpredictable and chaotic just like the environmental challenges to which the person is called to respond. A good modulation of vagal tone becomes indispensable for maintaining a good dynamic autonomic balance, as the imbalance from vagal inhibition is related to an increase in morbidity and mortality.[6] A simplification of HRV dynamics can result from system fatigue due to allostatic overload,[7] or to biological damage to system components, such as when heart failure leads to a reduction in entropy in heart rate.[8],[9],[10]

From the psychological point of view, loss of HRV is a consequence of emotional rigidity characterized by a tendency to respond to a wide range of situations with a stereotyped response: sadness, anxiety, suspiciousness, anger, and so on. However, not only is simplicity a hallmark of poor adaptation but also random variations to specific stimuli suggest a lack of modulatory control of responses. Both conditions are not favorable to a healthy aging and denote a decline in psychophysical resilience and an increase in the person's systemic vulnerability.

Psychophysiological systems with poor adaptability become particularly vulnerable to work, family and social stress, and subsequently to diseases. Chronic stress results in several patterns of deranged biochemical mechanisms of psychoneuroendocrineimmunology (PNEI), with an overall negative impact on health and aging.[11] Basically, HRV may mirror the psychological and biological consequences of distress, of PNEI dysregulation, and of all the related chronic degenerative diseases; hence, this parameter may represent a valuable tool in the assessment of aging processes which notoriously and progressively reduce HRV lifelong.[12],[13]

Of interest, HRV proved to be the most reliable bioparameter to assess human resilience,[14],[15] which is considered a key factor for a successful, healthy aging.[16] Thus, the assessment of HRV is nowadays considered a basic diagnostic test in aging medicine, as well as in several branches of biomedical sciences.

The improvement of a subject's resilience to any form of stress is the core target of hormesis,[17] which is a natural lifelong-persistent biological phenomenon characterized by a biphasic dose/response reaction; when hormesis pathways occur, a beneficial/healthy effect derives from the exposure to low/mild doses, and for a limited time, of an agent that is toxic or lethal at higher doses, or for higher durations.[17],[18] Through the repeated exposure to stressful events/stimuli, at low/mild doses and for a limited time, the human organism reacts building up by progressively developing an adaptation to stressful stimulations, which will finally result in a lower perturbation response over time, increasing the overall resilience.

An activation of several beneficial biochemical pathways has been shown in hormesis processes;[17],[18],[19] through the low/mild dose exposure to specific stressors (such as heat, cold, fasting, physical activity, polyphenols, and intellectual engagement), a greater adaptation (resilience) to the derangements of the balance of human homeodynamics is possible. More interestingly, a few studies have shown that these variations may be quantified and monitored through HRV.[14],[15]

Assessment of resilience, stress, and hormetic pathways through HRV[20] basically represents a useful opportunity in the overall management and follow-up of several prolongevity interventions.

Heart rate variability examination

The data-related to cardiac dynamics allow acquisition of a series of quantitative and qualitative information related to the state of the ANS.

Measurements under the time domain and those under the heart frequencies domain provide specific indicators with multiple valence. The indices under the time domain define the amount of variability in the interbeat interval measurements. The most useful, science-backed indices derived from HRV examination are as follows:

  1. Standard deviation of normal to normal (interbeat interval) (SDNN), which is measured in milliseconds, is a numerical expression of the action of the sympathetic and parasympathetic branches of the ANS
  2. Root mean square of the successive differences (RMSSD) is the mean square root of the differences of adjacent normal to normal intervals, numerical expression of the action of the parasympathetic system. A low value of this index indicates poor parasympathetic activity due to lack of recovery or high intellectual/emotional stress.

The power spectral analysis permits to separate HRV analysis in its constituent rhythms that operate within different frequency ranges. In the frequency domain, the most interesting data are retrieved during the 5 min session of free breathing, and they are related to the 3 zones of cardiac oscillation, each of which reflects specific activities of the ANS:

  1. Very low frequency (VLF) band: VLF includes oscillations between 0.0033 and 0.03 Hz, it represents the slowest changes in the heartbeat, and this range of frequencies is directly correlated with the activities of body thermoregulation and the hormonal cycle
  2. Low frequency (LF) band: LF includes oscillations between 0.03 and 0.15 Hz, it represents the slow changes in heart rate and is an index of sympathetic activity and of the effectiveness of the baroceptorial reflex, within the cardiovascular and respiratory systems' interaction
  3. High frequency (HF) band: HF includes oscillations between 0.15 and 0.40 Hz, it represents the fastest changes due to parasympathetic activity.

Finally, mean heart rate range (MHRR) represents another HRV analysis parameter, which indicates the difference between the maximum and the minimum heart rate during each breathing cycle. MHRR is expressed in beats per minute, as the mean of these heart rate differences for each measured cycle.[21],[22]

  Patients and Methods Top

An observational study was performed on 112 individuals who underwent HRV analysis, and the aim of the assessments was to detect possible variations of the baseline main HRV parameters/indexes after administration of different stressors/stimuli, at immediate/short-term follow-up.

The HRV examination was carried out by means of the device EmWave Pro Plus® (Quantum Intech, Inc. Boulder Creek, CA, USA), with data storage and analysis through a dedicated computer software. A pulse sensor was placed on the participant's earlobe (or fingertip in case of any ear alteration). The photoplethysmography technology is used to collect data from light reflection variations in the ear (or fingertip) capillaries as per heart cycle beats. Quantification of real-time HRV findings, both at rest for 5 min and during deep breathing test for 1 min, is based on the ability of hemoglobin to absorb light. The pulsation of blood flow in the capillaries reflects on the sensor absorbance of the light from the consequently variable hemoglobin content. Our targeted parameters during the examinations were SDNN, RMSSD, MHRR, mean heart rate (MHR), and the three frequency domain-based bands (VLF, LF, and HF).

The detection protocol was performed reproducing the same conditions and settings for each examination, with the individual comfortably sitting in a silent medical office, with closed eyes. First, a 1-min HRV deep breathing assessment was performed as follows: after a short acquaintance/try test, the individual was invited to follow the breath indicator, while using the diaphragm without forcing the action and focusing on this diaphragmatic movement. Six complete respiration cycles were performed in 1 min, 5 s inhalation and 5 s exhalation each cycle. Two minutes after the completion of this first part, the individual completed the second part of HRV assessment; he/she was invited to breathe normally and freely for 5 min more, leaving the mind thinking free.

Different cohorts of patients were investigated while undergoing 4 types of stimulations. The flowchart reported in [Figure 1] summarizes the 4 investigational protocols. More specifically: (a) one group of 62 individuals (32 females and 30 males) underwent HRV examination during basal normal/free breathing and during 1 min diaphragmatic/deep breathing of 10 s (5 s inhalation and 5 s exhalation); (b) the second group of 8 male individuals were examined in basal conditions and then they underwent HRV examination after one session of sauna exposure (90°C for 15 min) and/or cold exposure (cold shower) for 90 s; (c) the third group of 32 individuals (12 males and 20 females) took 2 tablets a day of a nutraceutical (Maqui 500®, Proeon) for one week; each tablet contained 500 mg of Maqui berry lyophilized extract, which is a polyphenol-rich food supplement, with 70.000 ORAC units (antioxidant power) per tablet. The HRV examination was performed in basal conditions before the nutraceutical sublingual intake, 75 min after the intake, and finally 7 days after; (d) the last group of individuals (10 males) performed one session of a standardized regimen of physical activity (50 min of mainly on-site isometric and isotonic exercising and stretching), and HRV examination was performed before the start of the exercising activity and immediately after the session.
Figure 1: Flowchart of the investigational heart rate variability protocol in the four cohorts of individuals

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The resulting data from the four cohorts undergoing HRV assessment were collected and stored in Microsoft Office Excel® files for the subsequent statistical analysis. From all the resulting figures, for each parameter, the mean value and standard deviation were calculated; P value was extrapolated from the comparison of all variables by means of t-paired test and Wilcoxon test; a 0.05 cutoff P value was considered as statistically significant for all the investigated parameters.

  Results Top

The outcomes of the HRV assessment in each of the four investigated groups are reported in [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]. All subjects underwent the two main steps of HRV analysis: 1 min diaphragmatic/deep and slow breathing and 5 min free breathing, both before and after the stimulatory stimulus (i.e., breathing, cold/heat, maqui nutraceutical intake and physical activity). No side effects were reported during the tests and the examinations.
Figure 2: Breathing. mean heart rate, standard deviation of normal to normal and root mean square of the successive difference values and P values after normal breathing for 5 min (blue color) and deep breathing for 1 min (red color)

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Figure 3: Maqui 500® intake. mean heart rate, mean heart rate range, standard deviation of normal to normal and root mean square of the successive difference investigation after deep breathing for 1 min, before taking Maqui 500® supplement (blue color), 75 min after the intake of the supplement (red color), 7 days after the intake of the supplement (yellow color)

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Figure 4: Maqui 500® intake. mean heart rate, standard deviation of normal to normal and root mean square of the successive difference investigation after normal breathing for 5 min, before taking Maqui 500® supplement (blue color), 75 min after the intake of the supplement (red color), 7 days after the intake of the supplement (yellow color)

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Figure 5: Maqui 500® intake: Very low frequency, low frequency and high frequency band investigation after normal breathing for 5 min, before taking Maqui 500® supplement (blue color), 75 min after the intake of the supplement (red color), 7 days after the intake of the supplement (yellow color)

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Figure 6: Physical activity: Mean heart rate, mean heart rate range, standard deviation of normal to normal and root mean square of the successive difference investigation after deep breathing for 1 min, before performing and after performing the physical activity (in blue and red color, respectively)

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In the cohort of the breathing assessment, we observed no significant difference (P = 0.66) as to the MHR value between basal/free and deep/slow breathing. Conversely both SDNN and RMSSD showed a statistically significant (P< 0.0001) remarkable increase of the mean value when passing from a free breathing to a deeper and slow breathing, shifting, respectively, from 41.18 to 59.71 and from 34.80 to 47.19. [Figure 2] summarises the main outcomes of breathing on HRV, highlighting the overall improvement of HRV parameters.

Thermal stimulation (sauna and cold shower)

Due to the small sample size of this specific cohort, we applied no statistical analysis to the resulting data. The [Appendix Figure 1] and [Appendix Figure 2], which summarize our findings derived from the HRV analysis after thermal stimulation, are reported in the appendix section; as overall, we got MHR increase and decrease immediately after sauna and after cold shower, respectively. Both SDNN and RMSSD significantly decreased (of more than 50%) after sauna, and the decrease was greater at the 1 min assessment. When combining sauna with immediately subsequent cold shower an overall increase of SDNN and of RMSSD was recorded. Lastly sauna sessions resulted in an immediate remarkable decrease of all frequencies (LF, VLF, HF), whereas cold shower after sauna induced a shift towards higher frequencies. Isolated cold shower noticeably increased SDNN and decreased RMSSD at 1 min assessment, returning close to the basal values after 5 min breathing.[INLINE: 1][INLINE: 2]

Maqui nutraceutical intake

The comparison among the findings of baseline (T0), 75 h minutes after sublingual intake of one tablet (T1) and 7 days after the regular intake of 2 tablets per day (T2) highlighted the following results: MHR had a relative decrease a T1, though it was not statistically significant (P = 0.1) whereas no change was detected at T2; MHRR conversely showed no change; at deep-breath 1 min assessment SDNN increased at T1 and T2 (from 46.30 to 56.82 and 54.15 respectively), but P = 0.10 in both cases [Figure 3]; RMSSD, instead, had a rise at T1 (from 37.54 to 47.18, P = 0.10), which was lower, at T2 [Figure 3]. During the free-breathing 5 min assessment a lower, but detectable, increase of SDNN and RMSSD (overall HRV increase) was recorded [Figure 4]. VLF, LF, and HF had an extremely individualized and scattered pattern, with great standard deviation of the mean value; however, HRV assessment showed a tendency towards a shift from lower to higher frequencies (parasympathetic activation) at T1 and especially at T2 [Figure 5].

Physical activity

HRV analysis before and just after one standardized exercising session is summarized in [Figure 6], [Figure 7], [Figure 8]; more in detail, MHR and MHRR showed no significant variation, whereas SDNN mildly increased at 1 min deep breath test, and it decreased at free breathing test. At 5 min free breathing check, RMSSD was slightly higher, but all these variations were not statistically significant; conversely VLF remarkably decreased (mean values from 241 to 139.48 P = 0.0281) and HF increased but the difference was not statistically significant.
Figure 7: Physical activity: Mean heart rate, standard deviation of normal to normal and root mean square of the successive difference investigation after normal breathing for 5 min, before performing and after performing the physical activity (in blue and red color, respectively)

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Figure 8: Physical activity: Very low frequency, low frequency and high frequency band investigation after normal breathing for 5 min, before performing and after performing the physical activity (in blue and red color, respectively)

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  Discussion Top

Health was defined as “the ability to adapt” in an editorial of “The Lancet” 10 years ago,[23] which confirmed the importance of coping, resilience and adaptation for a healthy aging; similarly aging brings human beings to experience a continuous dynamic process of responses to inner and environmental challenges, through the use of psychophysical resources. The greater the latter, the higher the subject's probability to have a healthy living and aging, as a greater resilience (hence a higher HRV) will indicate a more effective response to life-lasting challenges and return to balance. PNEI science clearly highlighted the strict (positive and negative) relationship between mind and body, the relevant importance of stress axis within the onset and the evolution of chronic degenerative diseases at all levels.[24],[25]

Engagement in daily tasks and stress involves the mobilization of the resources of the sympathetic system which, on reaching the goal, will withdraw to make way for the activities of the parasympathetic system. In the event that there is not a sufficient tonicity of one or the other system, or the prevalence of one, the performance of the tasks and the coping with stressful events could be ineffective and be inadequate for maintaining a state of health and well-being.

Excessive sympathetic activity, due especially to an incorrect and stressful lifestyle adopted by a wide segment of the population of more urbanized areas, is one of the causes of many disorders related to the lack of a balanced parasympathetic action (e.g., sleep dysregulation) or the depletion of resources (nutrient deficiency).

There is a range of oscillation of heart frequencies of great complexity and interest for the maintenance of the psychophysical well-being; the range of HRV power spectrum between 0.10 and 0.12 Hz is used in (vagus nerve targeting) biofeedback training as a reference range to obtain benefits in increasing the height of HRV.

Several studies in biomedical fields demonstrated that HRV assessment may provide highly reliable data on the overall balance between the sympathetic and parasympathetic system. The derived applications of this inexpensive, noninvasive and easy-to-use technique may be of significant help to assess several psychobiologic dysregulations; similarly, through HRV investigation, it may be possible to monitor a wide range of possible interventions aimed at improving individual resilience and more broadly the aging processes.

Literature data have clearly demonstrated how several stressors may activate hormetic pathways,[19],[26] as well as breathing and other biofeedback techniques showed a relevant action on the ANS (vagus nerve mainly).

Our cohort and observational study confirms the diagnostic value of HRV assessment and it highlights the immediate/short-term variations which are induced in most HRV parameters when applying a few of these stressors and techniques. The limited statistical significance of most variations of the resulting HRV parameters is obviously justified by the extremely short duration of our follow-up (as per the aim of this study, from few minutes to 7 days, as to the protocol for the different stressors). Notwithstanding this intentional early-detection approach, clear changes were elicited in several HRV parameters measurements after stressor application.

More specifically deep/slow breathing of just 1 min resulted in an extremely significant increase of SDNN and RMSSD values, which shifted from the lower part of the normal range towards the higher/average figures of sympathetic and parasympathetic tone. This confirmation of the extremely relevant action of slow breathing on HRV, enlightens once more the benefits of the wide range of different types of respiration. A few authors have repeatedly confirmed the close interaction between breathing and HRV, neurovegetative system and resilience ultimately.[27],[28],[29] From our limited experience and from literature data, we can speculate that a longer lasting deep/slow breathing session (e.g., 5 min minimum) would result in a higher increase of HRV as overall, especially as to the vagal component.

Our observational study on thermal (sauna and cold shower) stress proved the significant HRV variations (both sympathetic and parasympathetic system) induced by one single session at immediate term, with an interesting “compensatory” contribution of postsauna cold shower. These physiological adaptations from thermal stress have been reported by different studies by means of HRV for longer periods; more recently, a few Finnish authors elicited the beneficial effect of the sauna + cooling approach which increases HRV, reducing the sympathetic activity and increasing the parasympathetic one.[30]

Our experience with HRV assessment of polyphenol supplement intake (namely Maqui 500®) highlighted the reliability of this technique to investigate nutrients; we elicited an improvement of HRV sympathetic and parasympathetic branch parameters at immediate term (75 min after the sublingual tablet ingestion); however, this improvement did not reach the statistical significance (P = 0.10 in most cases). After 7 days of supplementation, HRV findings had a similar increase and we found a tendency (not statistically significant) of the three bands of frequencies to shift toward higher figures, which represents an expression of vagus nerve activation. Supplementation with maqui nutraceuticals is generally of longer duration and/or for repeated cycles and this polyphenol-rich berry proved of interest as to a few health biomarkers, as well as to psychophysical condition.[31],[32] Generally speaking, HRV assessment showed an interesting reliability in monitoring psychological, and physiological effects of nutrition and nutrients,[33] and our limited experience confirms this finding. The present data are suggestive of benefit, but we acknowledge that our results should be corroborated by larger and longer-lasting studies, to clarify, through HRV analysis, the possible healthy effects of maqui supplementation on the ANS.

Physical activity has a wide spectrum of evidence-based literature which highlights its beneficial effects on health and longevity; HRV reliably mirrors the beneficial variations induced by exercising in the ANS network.[34] Our immediate HRV assessment on a few subjects undergoing one session of a standardized form of physical activity, demonstrated a mild SDNN (sympathetic) increase, whereas some mild improvement of vagal activity (RMSSD) took place after 5 min. Interestingly, in this group of individuals, the variations in the frequency bands showed a clear shift in favor of the HF, with lower VLF figures, as a remark of the vagus nerve system activation.

The overall findings of our observational study show that immediate/short term HRV variations were significant with most stimulations which target ANS, hormesis, and psychophysical resilience; a longer duration protocol including similar stressors may represent a possible option to induce a progressive improvement of main HRV parameters, with beneficial repercussions on resilience and healthy aging ultimately.

In our experience, HRV assessment proved a reliable method to assess immediate/short-term sympathetic and parasympathetic system changes. Notwithstanding these promising preliminary results, one limitation of our study is represented by the very short time of investigation of the HRV variations generated by a few stimulations. In fact, our trial intentionally did not focus on the mid-/long-term beneficial/detrimental effects of specific stimuli on HRV, but conversely we investigated the HRV variations at very short-term follow-up. Repeated and longer duration HRV assessments of any interventions aimed at interacting with ANS may probably represent an option to follow-up their effects on psychophysical resilience, on the hormetic pathway, on PNEI and stress management.

Finally, we speculate that some of our statistical data are affected by the small sample size, hence larger cohorts would be required to have a more complete view of the HRV possibilities in monitoring the efficacy and safety of similar stressors.

  Conclusions Top

As overall, this observational study showed that HRV assessment was useful at immediate/short-term follow-up to detect the variations of a few parameters related to the ANS, in response to a few chemical and physical stimuli. In a few cases, statistically significant variations of the main parameters (e.g., SDNN, and RMSSD and frequency bands) were elicited after stimuli exposure. Studies including larger cohorts and longer follow-up are needed to corroborate our outcomes furthermore.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]

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