Effect of Acupuncture on Heart Rate Variability in Individuals With Multiple Sclerosis
Phase: NA
Conditions: Multiple Sclerosis
Sponsor: University of Sao Paulo

 
 
 
JHGD 2020, FOR PEER REVIEW 
http://revistas.marilia.unesp.br/index.php/jhgd/about/submissions 
EFFECT OF ACUPUNCTURE ON HEART RATE VARIABILITY IN INDIVIDUALS WITH 
MULTIPLE SCLEROSIS 
1. Introduction 
Multiple sclerosis (MS) is a severe autoimmune demyelinating disease that affects nervous system, has 
high morbidity and mortality and no effective targeted therapies are available. There are currently about 
3 million people who are affected by MS worldwide. The disease typically begins between the ages of 
20 and 50 and is twice as common in women as in men. (Lozano-Quilis et al., 2014). This equates to 1 
in 3,000 people in the world living with MS, and in countries with the highest prevalence as many as 1 
in every 300 people have MS (The Multiple Sclerosis International Federation, Atlas of MS, 3rd 
Edition, 2020). 
The correlations between clinical presentations and pathological changes are extremely complex 
(Obert, D. et al, 2016). The clinical subtypes of Multiple Sclerosis (MS) are determined to be: 
Relapsing-remitting, Secondarily Progressive and Primarily Progressive. These names were quickly 
incorporated into clinical practice, being used for better communication with the patient in relation to 
the course of the disease and to define specific populations in research. So far, no biomarkers or MRI 
signals have been identified which can distinguish between them. (Lublin, F.D, 2014; Lucchinetti, C., 
Bruck, W, 2004). 
Relapsing-Remitting type is defined by clinical outbreaks or increased lesions in the central nervous 
system (CNS), which generate major phenotypic changes. In turn, the Primarily Progressive type also 
shows alterations, generating lesions in the CNS, but there is no inflammatory activity, that is, outbreaks 
(Lublin, 2014; Lucchinetti and Bruck, 2004). Progression in this subtype occurs within months or years, 
with a deterioration process which occurs in a slowly and irreversibly way (Pender, M. P., 2004). The 
Secondarily Progressive type is an evolution of the RR phase, promoting a progressive and continuous 
neurological disability, and this transition mechanism is still poorly described (Trapp, B.D et al., 1999). 
One of the existing explanations is the scarcity of outbreaks in the RR phase with a poor response to 
corticosteroid treatments and a consequent increase in disabilities (Palace, J., 2003). 
Up to now there is no cure for MS, but there are several therapies aimed at improving function after an 
outbreak and preventing disability. Therapies, including medications and neuro rehabilitation can 
improve some symptoms, but cannot change the course of the disease (Lozano-Quilis et al., 2014). 

Autonomic impairment, including cardiovascular autonomic dysfunction, is not uncommon in patients 
with MS. Cardiovascular involvement may be expressed as poor physical fitness, fatigue, orthostatic 
hypotonia, or cardiac arrhythmias. The autonomic dysfunction observed in MS is believed to be 
associated with plaques located in the brainstem as well as spinal cord which affect autonomic areas 
and their connections (Cygankiewicz.and Zareba, 2013). 
Autonomic Nervous System (i.e. sympathetic and parasympathetic) is responsible for involuntary 
control of vital physiological functions, such as heart rate at rest and in response to stress. Therefore, 
adequate responses depend on the balance between the sympathetic and parasympathetic system (Tracy 
et al., 2016; Valenti et al, 2012; Vanderlei et al., 2009; Appelhans et al., 2006). 
To evaluate the activity of the ANS (Autonomic Nervous System) under physiological and pathological 
conditions, HRV measurement is a non-invasive technique capable, providing important information 
about the individual’s physical and mental health (Vanderlei et al., 2009; Gamelin et al., 2006). The 
presence of a high HRV represents efficient autonomic mechanisms, while low HRV may indicate 
abnormal adaptation of these mechanisms and insufficient ANS response (Khaled et al., 2006; 
Vanderlei et al., 2009), and it is a widely accepted means of assessing cardiac autonomic regulation 
(European Society of Cardiology and North American Society of Stimulation and Electrophysiology, 
1996). 
Some studies have documented abnormal HRV in patients with MS, and it showed a significant loss of 
vagal and increased sympathetic tone (Brezinova et al., 2004; Tombul et al., 2011). Dysautonomia in 
MS patients was more likely to occur in patients with a greater extent of physical disability (Merkelbach 
et al., 2001) and most pronounced in the progressive variant of MS disease (Zawadka-Kunikowska et 
al., 2020). In its turn Studer et al., (2017) and Shirbani et al., (2018) showed autonomic balance appears 
to be intimately linked with both the inflammatory activity of multiple sclerosis, which is featured by 
an overall hypoactivity of the sympathetic nervous system, and its compensatory plastic processes, 
which appear inefficient in case of worsening and progressive multiple sclerosis. 
Neuronal stimulation is a promising emerging field in modern medicine for the control of organ 
function and reestablishing physiological homeostasis during illness. Multiple recent studies show the 
potential of nerve stimulation to control inflammation and improve organ function in MS disorders. 
Acupuncture is a practice of Traditional Chinese Medicine and aims to both treat and prevent diseases 
through various methods, including the insertion of needles in specific points of the body, and should 
be analyzed as a complementary therapeutic measure in the treatment of MS. The location of the points 
used in Acupuncture is found in channels or medians through which the qi (energy) circulates, which 

in this objective way, aims to regulate the functions of the organism and allows the free flow of qi. The 
1997 National Institutes of Health consensus considers the use of acupuncture as a complementary 
therapy in the management of painful conditions (Stall et al., 2015).  
Besides that, neuromodulation with acupuncture can provide vital information about the functional 
organization of the nervous system (Ulloa, Quiroz-Gonzalez and Torres-Rosas, 2017). In this way 
Quispe-Cabanillas et al., (2012) evaluated thirty-one patients with Relapsing-Remitting Multiple 
Sclerosis undergoing treatment with immunomodulators, and they conclude that Electroacupuncture 
improved various aspects of quality of life, including a reduction in pain and depression. In addition, 
according to Anderson et al., (2014), evaluate the ability of acupuncture to modulate HRV could imply 
that acupuncture may function by regulating and integrating the various physiological systems in the 
body. In their review all randomized placebo-controlled trials (a total of 16) suggest that acupuncture 
improves HRV, but 12 of these studied healthy subjects, four studied subjects with medically diagnosed 
conditions, and none of these have studied these effects in people with MS. 
Given the above, the Objective of this study is to evaluate the heart rate variability in individuals with 
MS during the applicability of Acupuncture, to analyze the behavior of the autonomic nervous system 
before, during, and after therapy and the changes of the condition. It is necessary to emphasize the need 
for studies that prove the benefits of the adopted procedures, to be better understood and consequently 
better applied, expecting better results, and assisting rehabilitation professionals in choosing the 
therapeutic program based on scientific evidence. Considering the deliberations presented, as a 
hypothesis, we believe that individuals with MS will present greater sympathetic modulation that will 
tend to balance during and after the applicability of Acupuncture. 
2. Methods 
2.1. Trial Design 
A double-blinded randomized sham-controlled crossover trial with a 1:1 allocation ratio will be 
conducted, 40 individuals without previous illness will be evaluated, who will constitute the control 
group and 40 individuals with MS will constitute the experimental group, paired by age and gender. 
All participants will undertake active or sham acupuncture sessions. Groups A–S will start with 5 
sessions (1 per week) of active acupuncture combined with HRV evaluation for 20 min. After a 2-week 
washout, this group will be reallocated to another 5 sessions (1 per week) of sham acupuncture for 20 
min combined with HRV evaluation. Meanwhile, Group S–A will carry out the opposite protocol, 
participants will start an allocated 5 sessions (1 per week) of sham acupuncture combined with HRV 

evaluation, and after a 1-week washout period will be reallocated to 5 sessions (1 per week) of active 
acupuncture combined with HRV evaluation.  
As previously described by Shen et al. (2009), Tseng, Tseng and Chang (2015), Li et al. (2020) and Shi 
et al. (2013), a 2-week washout period has been used and was shown to be sufficient to reset the effects 
of the first 5 sessions. Figure 2 summarizes the planned experimental design. This research protocol 
follows the SPIRIT recommendations. 40 participants with MS will be recruited through referral by the 
coordinators of Brazilian multiple sclerosis association (ABEM), located in São Paulo state.  
Those interested in participating will undergo a detailed screening using the eligibility criteria for 
enrolment in the study. The sample size was calculated using statistical software (GPower 3.1.5) on the 
main outcome measure (i.e., the motor score). This calculation was based on data from one study with 
a group of individuals with CP who received tDCS (32). The power was 0.80; the alpha was 0.05; and 
the effect size was 0.65 (Cohen’s d). The sample estimation indicated that 30 participants would be 
necessary (i.e., 15 per group). With an adjustment to allow for a withdrawal rate (20%).  
This article has been reported in accordance with the Standard Protocol Items Recommendations for 
Interventional Trials (SPIRIT) (Chan et al., 2013a,b; Tables 1, 2). 
TABLE 1 | SPIRIT: description of the study protocol, schedule of enrolment, interventions, and 
assessments. 
 
S-A: Sham-Acupuncture; A-S: Acupuncture – Sham; EDSS: Expanded Disability Status Scale; MoCA: Montreal Cognitive 
Assessment; BRUMS: Brunel Mood Scale; BDI-II: Beck Depression Inventory Second Edition; EVA: Visual Analogue 
Scales. 
Enrolment Allocation
close-out
TIMEPOINT
t¹
0
t¹
t²
t³
t4
t5
t6
t7
t8
t9
t10
t11
1 day
7 day
14day
21 day 28 day 42 day 49 day 56 day 63 day 77 day 84 day
ENROLMENT
Eligibility screen
x
Informed consent
x
Assessment scales and tools
x
Allocations
x
INTERVENTIONS
Group S-A
Group A-S
ASSESSMENTS
EDSS
x
x
MoCA
x
x
BRUMS
x
x
x
x
BDI-II
x
x
EVA (pain)
x
x
x
x
x
x
x
x
x
x
PHYSIOLOGICAL ASSESSMENT
Ryodoraku
xx
xx
xx
xx
Thermography
xx
xx
xx
xx
Heart Rate Variability
x
x
x
x
x
x
x
x
x
x
Post-allocation
STUDY PERIOD

TABLE 2 | Trial characteristics based on WHO Trial Registration Data Set. 
DATA CATEGORY  
TRIAL INFORMATION 
PRIMARY REGISTRY AND TRIAL 
IDENTIFYING NUMBER 
ClinicalTrials.gov, ID:  
DATE OF REGISTRATION IN 
PRIMARY REGISTRY 
August 2022 
SECONDARY 
IDENTIFYING 
NUMBERS 
Ethical Committee of the University – January 18th, 2019  
CAAE: 01515118.3.0000.5505 
SOURCE(S) OF MONETARY OR 
MATERIAL SUPPORT 
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil 
(CAPES) 
PRIMARY SPONSOR  
Federal University of São Paulo– UNIFESP 
SECONDARY SPONSOR(S)  
NA 
CONTACT FOR PUBLIC QUERIES 
TDS, CF 
CONTACT 
FOR 
SCIENTIFIC 
QUERIES 
TDS, CF 
PUBLIC TITLE 
EFFECT OF ACUPUNCTURE ON HEART RATE VARIABILITY OF 
PEOPLE WITH MULTIPLE SCLEROSIS: A STUDY PROTOCOL FOR A 
DOUBLE-BLINDED RANDOMIZED CONTROLLED TRIAL 
SCIENTIFIC TITLE 
ANALYSIS OF AUTONIMIC CARDIAC MODULATION DURING 
ACUPUNCTURE IN INDIVIDUALS WITH MULTIPLE SCLEROSIS. 
COUNTRY OF RECRUITMENT 
Brazil 
HEALTH 
CONDITION(S) 
OR 
PROBLEM(S) STUDIED 
Multiple sclerosis 

INTERVENTIONS  
For data collection, the following instrument will be used: Visual Analogue Scale 
of Pain. This scale will be applied at the beginning of each intervention day. In 
sequence, the participants will perform the Thermography protocol, which 
comprises capturing images in various postures in the orthostatic position (in the 
inicial and final assessments).   
Afterwards, the participants will remain in the supine dorsal position on a 
stretcher at rest for 10 minutes to start the SNA analysis with the Ryodoraku 
(device that measures the electroconductivity of certain points on the skin) by the 
digital reading of the 24 points called ryodos (SCILIPOTI, 2007). 
Continuing the procedure, immediately after the placement of the Polar V800, an 
instrument for analyzing HRV, the placing of needles at the points carefully 
selected for this research begins. The needling will proceed for the experimental 
group using specific Traditional Chinese Acupuncture points: E36, BP6, F3, IG4, 
for 20 minutes while the control group will receive needling in sham points 
(points not recognized as Acupuncture points), with the duration of 20 minutes.  
Final phase, new reading with Ryodoraku and Thermography. 
KEY 
INCLUSION 
AND 
EXCLUSION CRITERIA 
Inclusion Criteria 
This study will include individuals diagnosed with MS, aged over 18 years, with 
motor and intellectual capacity to understand the evaluations, with light and 
moderate functional classification levels (EDSS scale) and who accept to 
participate in the study through agreement with informed consent form. 
Exclusion Criteria 
Participants will be excluded if they (1) do not understand the evaluations; (2) 
cardiac diseases that impede the assessment of HRV; (3) have a cardiac 
pacemaker. 
STUDY TYPE INTERVENTIONAL 
ALLOCATION:  
Randomized 
MASKING:  
Double-blind 
ASSIGNMENT:  
Crossover 
PRIMARY PURPOSE:  
Treatment 

DATE OF FIRST ENROLMENT  
September 2021 
TARGET SAMPLE SIZE  
80 participants 
RECRUITMENT STATUS  
Recruiting 
PRIMARY OUTCOME(S)  
Heart Rate Variability improvement 
KEY SECONDARY OUTCOME(S) 
Ryodoraku and Thermography improvement 
 
2.2. Study Location and Period 
Randomization 
To perform the allocation procedure, the encoded groups will be placed inside a closed opaque 
envelope, which will be labeled with the code for each participant. Envelopes will be opened only 
during the first time of acupuncture or sham intervention.  
The results assessors to are blinded, will receive results without group description.  
2.3. Study Population and Eligibility Criteria 
Inclusion Criteria 
This study will include individuals diagnosed with MS, aged over 18 years, with motor and intellectual 
capacity to understand the evaluations, with light and moderate functional classification levels (EDSS 
scale) and who accept to participate in the study through agreement with informed consent form.  
Exclusion Criteria 
Participants will be excluded if they (1) do not understand the evaluations; (2) cardiac diseases that 
impede the assessment of HRV; (3) have a cardiac pacemaker. 
Withdrawal Criteria 
Participants will be withdrawn from the study if they are not willing to continue, cannot be present on 
the day of the experiment, or miss one treatment sessions out of 10. 

Screening tools 
Functional assessments 
Expanded Disability Status Scale (EDSS) 
The Expanded Disability Status Scale was developed by Kurtzke (1983). It is an assessment scale 
evaluating the functional systems of the central nervous system, it is used to describe disease 
progression in patients with MS and to assess the effectiveness of therapeutic interventions in 
clinical trials. It is subdivided into eight functional systems: pyramidal, cerebellar, brainstem, 
sensitive, bladder, intestinal, visual and mental functions. Confirmation of EDSS increase at 12 or 
24weeks or a later time point reduces the likelihood of capturing events that may subsequently 
revert (Kappos et al., 2017). It consists of ordinal rating system ranging from 0 (normal neurological 
status) to 10 (death due to MS) in 0.5 increments interval (when reaching EDSS 1). The lower scale 
values of the EDSS measure impairments based on the neurological examination, while the upper 
range of the scale (> EDSS 6) measures handicaps of patients with MS. The determination of EDSS 
4 – 6 is heavily dependent on aspects of walking ability (Meyer-Moock et al., 2014). 
Cognitive assessments 
Montreal Cognitive Assessment, MoCA 
The MoCA is a screening test, useful tool for the detection of mild dementia or mild cognitive 
impairment. Its cover 10 cognitive domains using rapid, sensitive, and easy-to-administer cognitive 
tasks. (Smith & Holmes, 2007). It is a 10-minute test, in there patients answer items with the 
following : the short-term memory recall task, Visuospatial abilities, a three-dimensional cube, 
Executive functions, a phonemic fluency task, a verbal abstraction task, sustained attention task, a 
serial subtraction task, and digits forward and backward, language, orientation to time and place 
(Nasreddine, 2005). Each domain generates a different score, totaling 30-point cognitive screening 
test designed in a one-page, the suggested cut-off point on the MoCA is 26 (Smith & Holmes, 
2007). 
 
Emotional and behavior assessments 
Brunel Mood Scale (BRUMS) 
The Brunel Mood Scale was developed to allow a quick measurement of the mood state of 
populations composed by adults and teenagers. It consists of 24 items to assess mood change in the 
six mood dimensions - tension, depression, anger, vigor, fatigue, and confusion. The question used 
is “How do you feel now”. The items are assessed on a 5-point scale anchored from “not at all” (0) 

to “extremely” (4). Each subscale contains four items. With the sum of the responses of each 
subscale, a score can be obtained that can vary from 0 to 16. The scale takes about one to two 
minutes to be answered (Rohlfs et al., 2008), it was presented as a tool for detection of the over-
training syndrome, besides that it has been used in different populations (Silva et al., 2021) and 
contexts in Brazil (Brandt et al., 2016) and other countries (Lan et al., 2012). Including for 
evaluations in populations with cardiac disorders (Sties et al., 2014). 
 
Beck Depression Inventory Second Edition (BDI-II) 
The BDI-II is a relevant psychometric instrument, showing high reliability, and capacity to 
discriminate between depressed and non-depressed subjects. (Wang & Gorenstein, 2013). The main 
factors of the inventory are a cognitive and a somatic factor, and that some items can shift between 
factors depending on which population samples represent (Arnarson et al., 2008). Examples of 
these items include questions regarding changes in sleep patterns, difficulty concentrating, sadness, 
self-dislike, crying, loss of energy, and suicidal thoughts (Strunk & Lane, 2016). BDI-II is a self-
report measure of depression. Twenty-one symptoms of depression are rated on a 4-point scale (0 
to 3) scale, within the time frame of the past 2 weeks. The total scores are obtained by summing 
the ratings for all items. Scores ranging between 0 and 13 are indicative of minimal depression; 
scores that fall between 14 and 19 are considered to reflect a mild level of depression; scores of 20 
to 28 are considered moderate; and a score ranging from 29 to 63 is labeled severe (Dozois, 2010). 
 
Evaluation analysis tools 
Visual Analogue Scales (VAS) 
The VAS is by far the most frequently used assessment instrument (Reed e Van Nostran, 2014). 
Visual analogue scales are psychometric measuring instruments designed to document the 
characteristics of disease-related symptom severity in individual patients and use this to achieve a 
rapid (statistically measurable and reproducible) classification of symptom severity and disease 
control (Klimek et al., 2017). In our study, we will use it for pain mensures. It consisted of 100 mm 
lines giving a score ranging from 0 up to 10, whose endpoints were designated as ‘no pain’ and’ 
the worst pain imaginable’. Although some pain sensations may be equally intense and unpleasant, 
the patient need to judge the two aspects independently, to indicate the relative intensity of your 
pain sensation: the higher number, the greater the intensity (Heller, Manuguerra and Chow, 2016). 
 

Blinding 
The participants and assessors of results analysis will remain blind to group allocation during the 
study. To ensure proper blinding, participants will receive the same care, but with points outside 
the area of the correct acupuncture points.  Considering that the needle applicator must be a 
qualified professional, it is not possible that he is blind. 
 
 
Intervention 
For data collection, the following instrument VAS Scale will be used. This scale will be applied at the 
beginning of the data collection. In sequence, the participants will perform the Thermography protocol, 
which comprises capturing images in various postures in the orthostatic position.   
Afterwards, the participants will remain in the supine dorsal position on a stretcher at rest for 10 minutes 
to start the SNA analysis with the Ryodoraku (device that measures the electroconductivity of certain 
points on the skin) by the digital reading of the 24 points called ryodos (SCILIPOTI, 2007). 
Continuing the procedure, immediately after the placement of the Polar V800, an instrument for  
analyzing heart rate variability, the placing of needles at the points carefully selected for this 
research begins. The needling will proceed for the experimental group using specific Traditional 
Chinese Acupuncture points: E36, BP6, F3, IG4, for 20 minutes while the control group will receive 
needling in sham points (points not recognized as Acupuncture points), with the duration of 20 
minutes.  Final phase, new reading with Ryodoraku and Thermography. 
 
Ryodoraku 
Based on the Ryodoraku (meridian) theory, developed by Dr. Yoshio Nakatani (Nakatani, 1956), 
there are 12 meridians on the right and left side of the human body respectively. The property of 
Ryodoraku can reflect the condition of certain organ(s) by analyzing and comparing their mutual 
relations and changes with micro-electrical current. The electrical state of the acupuncture points 
of the human subject is measured by a computerized testing instrument with a very low electrical 
current. From the traditional theory, Ryodoraku is a pathological phenomenon. The mechanism can 
be explained by the viscera-skin sympathetic nerve reflex. The impulses from the viscera radiate to 
the spinal cord. The reflex zones are then reflected onto the skin surface via the efferent sympathetic 
nerves and appear as a longitudinal connecting system, just like meridian lines (Weng et al., 2004). 

Electrodermal screening measures either skin impedance or conductance at acupuncture points as 
diagnostic aids for planning treatment strategies. Skin impedance is the skin’s opposition to the 
flow of current. The practice of measuring skin impedance is based on the widely held assumption 
that acupuncture points are loci of decreased impedance compared to skin sites where there are no 
known acupuncture points (Pearson et al., 2007). 
Recently, researchers have begun to explore it at acupoints as a potential physiological outcome 
measure for use in clinical trials of acupuncture. The technique consists of measuring electrical 
conductance at representative points for each meridian, generally the Yuan points on the wrists and 
ankles. Because of the high variability in skin conductance measurements, readings are never taken 
as absolute but rather as relative to all other measurements on the same patient and reported on a 
normalized scale. Increased conductance (decreased resistance) is believed to represent an excess 
in the meridian being measured, and decreased conductance (increased resistance) represents a 
deficiency (Colbert et al., 2011). 
Thermography 
Infrared cameras measure the wavelength of infrared radiation an object emits and converts it to 
electrical energy, which is then used to measure surface temperature. As an imaging technology, 
has numerous advantages resulting from its non-invasive and non-contact approach. In this way, 
thermal information can be assessed from a distance, a valuable feature for field biology, without 
the need for surgical interventions or handling that have prolonged effects on body temperature 
(Tattersall, 2016). It is one of the most promising methods of probing the psychological status of 
human beings (Hong e Hong, 2015). 
The human skin acts as a black-body radiator operating at the body temperature generating an 
infrared radiation spectrum with a broad peak at around = 9.5um. The body’s core temperature is 
developed in the central part of the body where the major organs are located (heart, lungs, liver, 
etc.) and heat is transported by the blood through the arteries to the rest of the body. Because the 
environment temperature is usually lower than the core temperature (37 °C), there is a cooling 
effect from the skin to the environment and heat transfer from the blood into the surrounding tissue 
and to the environment takes place (De Graaf et al., 2019). 
With the aid of thermometry infrared electromagnetic waves of 0.7–1000 M (which the human eye 
is unable to identify) can be visualized, healthy human bodies emit waves with a length of 3–10 M. 
Temperature differences as low as 0.1°C can be pursued and may help track response to 
acupuncture as opposed to needling of non-acupoints to objectively estimate effects of needling 
(Agarwal-Kozlowski, Lange e Beck, 2009). 

Lastly, changes in temperature detected will be related to changes in blood flow when the external 
environment is stable (no changes in irradiative, conductive, or convective heat transfer), and thus 
sensitive to acute changes in sympathetic activity which would induce vasoconstriction as the 
vasculature is not parasympathetically innervated. This temperature rise under extreme stimuli may be 
due to increases in cardiac output, which may increase perfusion pressure or the release of local factors 
such as nitric oxide from nearby endothelial cells, and it may be sympathetically mediated but could 
relate to parasympathetic withdrawal that induces cardiac acceleration, suggesting a complex interplay 
of autonomic control (Huggins e Rakobowchuk, 2019). 
 
Analysis of Heart Rate Variability 
After the initial evaluation, the capture strap will be placed on the volunteers’ chests, and the heart 
rate receiver (V800, Polar) will be placed on their wrists, previously validated equipment for 
capturing the heart rate, beat by beat, and the use of their data for HRV analysis (GAMELIN et al., 
2006; KINGSLEY et al., 2005). 
After placing the strap and the monitor, the individuals will be positioned in a sitting position and 
will remain at rest, breathing spontaneously for 20 minutes. Following that, the analysis will be 
made during the performance of Acupuncture.  
or analysis of HRV data in the sitting position, 1000 consecutive RR intervals will be used, and 
during the tasks, 256 consecutive intervals will be used. Then, a manual filtering will be performed 
through the Microsoft Excel program, to eliminate premature ectopic beats and artifacts, and there 
will be no substitution, only the elimination of artifacts. After manual filtering, the first 1000 
beating intervals are chosen and only series with more than 95% of sinus beats will be included in 
the study (GODOY et al., 2005). The software used to assess HRV will be the Kubios HRV.  
HRV analysis will be performed using linear methods (time and frequency domains) and non-linear 
methods that will be analyzed using the softwares: HRV analysis, CDA_Pro. 
 
Linear Methods (Time and Frequency domain)  
 
In the time domain, the RMSSD, pNN50 and SDNN indexes will be used. The RMSSD which is 
Square root of the mean of squared differences between successive beat intervals (Marães et al., 
2003; Batista et al., 2019). The pNN50 index, in turn, is a sensitive and easily interpretable marker 

of the parasympathetic SNA modulation, defined as the percentage of successive differences in the 
R-R interval whose absolute value exceeds 50ms. SDNN, on the other hand, reflects the participation of 
both branches of the SNA and corresponds to the standard-deviation of the average of all normal RR intervals, 
expressed in ms (Pumprla et al., 2002; Aubert et al., 2003; Bittencourt et al., 2005; Ribeiro e Moraes 
Filho, 2005). 
For the analysis of HRV in the frequency domain, low frequency spectral components (LF – range 
between 0.04 to 0.15 Hertz) will be used in absolute units and high Frequency (HF – range from 
0.15 to 0.4Hertz), in normalized units, and the ration between these components (LF/HF), which 
represents the relative value of each spectral component in relation to the total power, minus the 
exceptionally low frequency components (VLF). The algorithm used for the spectral analysis will 
be the fast Fourier transform - FFT (256 s window with 50% overlap) (Vanderlei et al., 2009; 
Martinelli et al., 2020; Carvalho, 2009). 
 
Nonlinear Methods (Fluctuation Analysis, Depuration of Trends and Poincaré Graph)  
 
For HRV analysis using nonlinear methods, the DFA quantifies the presence or absence of fractal 
correlation properties of RR intervals and has been validated by time series data. This method 
calculates the fluctuation of the mean square root of the integral and depurates the time series, 
allowing the detection of intrinsic self-similarity embedded in the non-stationary time series. The 
DFA graph is not strictly linear, but consists of two distinct regions of distinct curves, separated at 
one point, suggesting that there is a short-term factal scale exponent (a1) during periods of 4-11 
beats (or 4 to 13), and a long-term exponent (a2), for longer periods (greater than 11 beats) 
(Carvalho et al, 2009; Moraes et al., 2019; Godoy et al., 2007). 
The Poincaré graph is a quantitative method of analysis, based on the changes in the sympathetic 
or parasympathetic modulation of the heart rate over the subsequent intervals, without the need for 
the property of data stationarity (Godoy et al., 2007; Guerra et al., 2019). The following indexes 
will be calculated: SD1 (standard deviation of instantaneous beat-to-beat variability in the short 
term), SD2 (long-term standard deviations of continuous R-R intervals) and the SD1/SD2 ratio 
(BRUNETTO et al., 2005). 
 
Primary Outcome 
 

We will observe changes in the ANSs after intervention with active acupuncture and sham 
acupuncture in patients with MS, during a period of 28 days and after 2-weeks follow-up, following 
by more 28 days of intervention. 
 
Secondary Outcome 
 
We will analyze changes in Ryodoraku and Thermography regarding both interventions (active and 
sham acupuncture). 
 
DATA ANALYSIS 
 
If the data meet the assumptions for using parametric analysis, analysis of variance (ANOVA) will 
be performed to identify intra and intergroup differences. The differences, if any, will be detected 
by Bonferroni’s post-hoc test. If the assumptions of ANOVA are not met, non-parametric analyses 
will be used to identify and locate the differences: Friedman -post-hoc Wilcoxon as test (intra-
group) and Kruskal-Wallis and U of Mann-Whitney as post-hoc test (intergroups).  A significant 
level of 0,05 (5%) will be defined and all intervals built along the work will be with e 95% statistical 
confidence. The statistical program will be the SPSS (Statistical Package for Social Sciences),                     
version 11.0 (Frankfort-Nachmias and Leon-Guerrero, 2005). 
 
 
RISKS AND BENEFITS  
 
The research is based on analyzing the variability of SNA – Autonomous Nervous System with the 
therapeutic use of an Acupuncture protocol and a complementary protocol of Thermography in 
individuals with Multiple Sclerosis. Therefore, it is considered research with human beings and as 
such has the duty to safeguard the integrity of those involved. This safeguard will be in relation to 
the questions of privacy, secrecy, and the minimization of any discomfort.  
Risks 

The participation in this research does not bring complications. The risks in their participation in 
the research are minimal and related to the use of acupuncture needles, which are sterile, of single 
and individual use, discarded at the end of the procedure in a compartment suitable for sharp 
perforating materials. Related to the use of the camera, the participant does not adapt with the 
environment acclimatization.  
And regarding the use of Ryodoraku – Model NKL, the risks are also minimal, because there may 
be a slight discomfort in relation to the pressure of the tip with wet cotton on the skin applied on 
the points to be evaluated during the reading. Therefore, there will always be a responsible person, 
attentive in the orientations to the participant.  
The procedures adopted in this research obey the Criteria of Ethics in Research with Human Beings 
according to resolution 44/12 of the National Health Council (Conselho Nacional de Saúde). None 
of the procedures used puts their dignity at risk.  
Confidentiality: all information that will be collected is strictly confidential, being restricted only 
to the researcher (a) and the supervisor (a). Only they have the knowledge of the data collected for 
the development of the research,  
Benefits 
Research participants will have indirect benefits. They will benefit from future information that 
may improve the quality of life of the participants. Therefore, there is no immediate benefit 
foreseen to the participants of this research, only after the conclusions of this work that can 
contribute in a significant way to the adequate, organized and effective elaboration of the 
diagnostic methods and treatment in Acupuncture.  
Corroborating, we hope that this study will contribute significantly to the rehabilitation programs, 
so that knowledge observed and acquired from this research can contribute to the advancement of 
science and health of these patients. The researcher compromisses to divulge the obtained results.   
 
Trial status  
Participant recruitment will start in September 2022, it was stopped due to quarantine in March 
2020. Expected to end in May 2023. Study completion is estimated by December 2022. 
 
 

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