Multiple sclerosis (MS) is a chronic autoimmune central nervous system disease characterized by inflammation, demyelination, and neurodegeneration, resulting in motor, cognitive, speech-language, psychological, and cardiac autonomic impairments.

This double-blind, randomized, controlled crossover trial investigates the effects of repetitive transcranial magnetic stimulation (rTMS) applied over the primary motor cortex (Cz) and left dorsolateral prefrontal cortex (F3), combined with virtual reality (VR)-based exercise (MoveHero platform) and conventional physiotherapy, in 30 individuals with MS (EDSS 1.0-6.5).

Participants are randomized to Active-then-Sham or Sham-then-Active sequence groups. Each phase consists of 10 consecutive weekday rTMS sessions, followed by at least four weeks washout before crossover. Both targets use a circular coil at 10 Hz. Cz is stimulated at 90% resting motor threshold (30 trains, 50 pulses/train) and F3 at 110% resting motor threshold (40 trains, 50 pulses/train). The sham condition replicates the procedure with the coil inverted at 10% intensity. On six of ten intervention days, rTMS is followed by VR exercise and balance-focused physiotherapy.

Outcomes assessed at baseline and post-intervention include cardiac autonomic modulation (heart rate variability), balance, functional mobility, manual dexterity, fatigue, quality of life, depression, attention, executive function, working memory, psychological well-being, verbal fluency, and speech-language function (aphasia, dysphagia, dysarthria, voice quality). Follow-up reassessments are conducted at six months and one year.

**Background and Rationale** Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system characterized by inflammation, demyelination, gliosis, and neuronal loss. Lesions are restricted to the central nervous system, sparing the peripheral nervous system. Pathological mechanisms include perivascular lymphocytic infiltration and macrophage-mediated myelin degradation, predominantly during relapses, as well as gliosis and neurodegeneration associated with disease progression. MS typically manifests between ages 20 and 50 and is twice as common in women.

Neurological symptoms vary according to lesion location and include visual impairment, sensory disturbances, focal weakness, urinary and bowel dysfunction, and cognitive impairment, all of which impact gait, balance, and functional mobility. Although no cure exists, therapeutic strategies targeting post-relapse recovery, relapse prevention, and disability reduction have been developed. With disease progression, individuals become increasingly dependent for activities of daily living.

Beyond motor and cognitive impairment, autonomic nervous system (ANS) dysfunction has been documented in MS, manifesting as orthostatic intolerance, postural tachycardia syndrome, and reduced heart rate variability (HRV). These conditions are related to brainstem lesions and reflect dysregulation between sympathetic and parasympathetic activity at the sinoatrial node. HRV, a non-invasive index of cardiac autonomic modulation, captures beat-to-beat variation in RR intervals and provides prognostic information regarding cardiovascular and autonomic health.

Virtual reality (VR)-based rehabilitation has demonstrated benefits for sensorimotor learning, balance, strength, cognition, and attention in neurological populations, including MS. The MoveHero platform, developed by the Research and Technological Application in Rehabilitation Group (PATER) at the University of São Paulo, is a task-oriented VR game requiring timed upper or lower limb movements to targets presented on screen, with visual and auditory feedback.

Non-invasive brain stimulation, particularly repetitive transcranial magnetic stimulation (rTMS), has emerged as a promising neuromodulatory approach in neurological and neuropsychiatric conditions. rTMS modulates neural excitability through time-varying magnetic fields that induce electrical currents in cortical tissue, promoting neuroplasticity. Evidence supports its efficacy for depression, aphasia, spasticity, fatigue, and cognitive impairment in MS. However, no study to date has examined the combination of rTMS with VR exercise and conventional physiotherapy targeting motor, cognitive, speech-language, psychological, and cardiac autonomic outcomes concurrently in individuals with MS.

--- **Study Design**

This is a triple-blind, randomized, controlled crossover trial. Participants will be randomized in a 1:1 ratio to one of two sequence groups: Active-then-Sham (A-S) or Sham-then-Active (S-A), using an online randomization tool. Allocation will be managed by a blinded investigator who is the sole custodian of the randomization file. Group homogeneity at baseline will be verified for age and functional status (EDSS); if groups are not comparable, rerandomization will be performed.

Participants, outcome assessors, physiotherapy and VR intervention administrators, and the statistician will remain blinded to group allocation throughout the study. Only the randomization manager and rTMS operators will have access to allocation information.

-- **Participants**

Thirty individuals with a confirmed diagnosis of MS, of both sexes, aged 20 to 70 years, with EDSS scores between 1.0 and 6.5 will be recruited from the Brazilian Multiple Sclerosis Association (ABEM) in São Paulo, Brazil. All participants will continue their routine activities at ABEM (physiotherapy, speech-language therapy, and psychotherapy) throughout the study period, with rTMS and VR sessions added to their existing schedule.

Exclusion criteria include: cranial metallic implants or skull deformities, cardiac arrhythmias or atrioventricular block, congenital anomalies (including congenital heart disease or pulmonary malformations), history of seizures, use of medications interfering with ANS function (such as antiarrhythmics or insulin), and HRV recording artifact rates exceeding 5%. Participants will be withdrawn if they miss assessment days (Day 1 or Day 10), accumulate three or more absences, are unable to perform the required exercises, cannot tolerate rTMS, fail to complete questionnaires, or withdraw consent. Cardiovascular events during participation will also result in withdrawal.

Sample size was calculated assuming alpha of 5%, beta of 20% (power = 80%), and a 10% between-group difference in VR motor performance, yielding 30 participants.

-- **Intervention**

Each intervention phase consists of 10 consecutive weekday sessions (Monday to Friday, two weeks). Following Phase 1, a washout period of at least four weeks is observed before participants cross over to the opposite condition for a further 10 sessions.

-- **1. rTMS Protocol**

On Day 1, target sites are localized using the 10-20 EEG system with a measuring tape, and the motor hotspot is identified as the scalp position eliciting maximum contralateral hand muscle contraction. The resting motor threshold (RMT) is determined for each participant individually.

In the active condition, rTMS is applied using a circular coil at the primary motor cortex (Cz) with the following parameters: 10 Hz frequency, 50 pulses per train, 30 trains, 20-second inter-train interval, at 90% RMT. Subsequently, rTMS is applied using a circular coil at the left dorsolateral prefrontal cortex (F3) with the following parameters: 10 Hz frequency, 50 pulses per train, 40 trains, 20-second inter-train interval, at 110% RMT.

In the sham condition, the same procedure is performed at identical sites, but with the coil inverted and machine intensity set at 10%, precluding cortical stimulation while maintaining procedural appearance. Each participant retains their individually labeled cap across all sessions to ensure consistent coil positioning. The procedure is administered by an experienced operator to ensure participant blinding to allocation.

At the end of the full protocol, an adverse effects questionnaire will be administered to all participants.

-- **2. VR and Physiotherapy Protocol**

On Days 1, 2, 4, 6, 7, and 9 of each phase, rTMS is followed immediately by VR exercise using the MoveHero platform and conventional physiotherapy. On remaining days (Days 3, 5, 8), rTMS is administered alone.

The MoveHero VR protocol targets lower limb function. Participants perform three game sessions in which colored balls fall on the screen at a constant rate, and the participant must reach the corresponding target with timed lower limb movements. Background music is used to enhance engagement but does not determine task timing. Correct movements are reinforced with green visual feedback and point scoring; errors trigger red visual and auditory feedback. Difficulty increases automatically with accumulated score. Sessions are conducted three times per week.

The physiotherapy protocol consists of a structured static and dynamic balance training circuit performed immediately after rTMS, three times per week. The circuit includes: walking, squat on unstable surface, static balance with destabilizers at the base of support, stair ascent and descent training, and lateral gait exercises.

-- **Outcome Assessments**

Assessments are conducted at Day 1 (baseline) and Day 10 (post-intervention) of each phase, with follow-up reassessment of all physiotherapy, speech-language, and psychology instruments at six months and one year after completion of the full crossover protocol.

-- **1. Cardiorespiratory Variables**

Systolic and diastolic blood pressure (aneroid sphygmomanometer, BIC; stethoscope, Littmann Lightweight II S.E.), heart rate, respiratory rate (counted over 60 seconds without participant awareness), and peripheral oxygen saturation (pulse oximeter, G-tech) are measured before each session component and at the end of data collection.

-- **2. Heart Rate Variability**

HRV is recorded on Days 1 and 10 of each phase using a chest-worn heart rate monitor (Polar V800, previously validated for beat-to-beat RR interval capture), with data stored via the Elite HRV application and transferred to computer. Participants remain seated in a comfortable recliner for 15 minutes at rest, breathing spontaneously, without speaking, sleeping, or making abrupt movements. HRV is also recorded during rTMS application at both Cz and F3 cortical targets.

Analysis uses 1000 consecutive RR intervals. Manual filtering is performed in Microsoft Excel to exclude ectopic beats and artifacts, without data substitution. Only recordings with greater than 95% sinus beats are included. HRV analysis is performed using Kubios HRV software, encompassing time-domain indices (RMSSD, SDNN, pNN50), frequency-domain indices (LF, HF, LF/HF ratio), and nonlinear indices.

-- **3. Physiotherapy Outcomes**

Fatigue: Modified Fatigue Impact Scale (MFIS, 21 items, three domains: physical, cognitive, psychosocial; scores below 38 indicate absence of fatigue).

Quality of life: Multiple Sclerosis Quality of Life Scale (MSQOL-54). Balance: Berg Balance Scale (14 items, maximum score 56). Functional mobility: Timed Up and Go Test (TUG; best of three trials recorded). Manual dexterity: Box and Block Test (dominant and non-dominant hand, blocks transferred in 60 seconds).

-- **4. Speech-Language Outcomes**

Cognitive-linguistic screening: Mini-Mental State Examination (MMSE), with aphasia subtype characterization based on fluency, comprehension, naming, and repetition profile.

Dysphagia: Dysphagia in Multiple Sclerosis Questionnaire (DYMUS-BR, 10 items). Voice-related quality of life: Voice-Related Quality of Life questionnaire (VQoL-V/QVV, 10 items, physical and socioemotional domains).

Dysarthria: Living with Dysarthria questionnaire (VcD, 50 items across 10 sections).

-- **5. Psychological Outcomes**

Depression: Beck Depression Inventory-II (BDI-II, 21 items; categories: minimal, mild, moderate, severe).

Attention: Psychological Battery for Attention Assessment (BPA; concentrated, divided, and alternating attention subtests, plus General Attention index).

Executive function and processing speed: Five Digit Test (FDT; Reading, Counting, Choice, and Alternation tasks; Inhibition and Flexibility indices).

Working memory: WAIS-III Digit Span subtest (forward and backward). Psychological well-being: Flourishing Scale (FS, 8 items, Likert 1-7). Verbal fluency: Phonemic fluency (FAS/CFL letters, one minute per letter, three trials) and categorical fluency (Animals/Fruits, one minute per category).

A sociodemographic questionnaire is completed at baseline, covering age, education, marital status, employment status, MS type, EDSS, disease duration, last relapse, medication use, symptom profile, mobility aids, hand dominance, computer experience, sleep quality, visual and cognitive problems, and family history of MS.

-- **Statistical Analysis**

Pre- and post-intervention data will be compared within and between active and sham conditions across all outcome domains. Pearson correlation coefficients will be used to examine associations between HRV indices, VR motor performance, and sociodemographic and clinical variables (age, EDSS, disease duration, sex, employment status). A significance level of 0.05 and 95% confidence intervals will be adopted throughout. All analyses will be performed using SPSS version 26.0.

Inclusion Criteria:

• Participants with a confirmed diagnosis of Multiple Sclerosis.
• Ability to understand and follow simple instructions and to participate in the experimental procedures.
• Expanded Disability Status Scale (EDSS) score ≤ 6.5.
• Ability to maintain upright standing position (with or without assistive device) and perform basic static and dynamic balance tasks required for the intervention.

Exclusion Criteria:

• Presence of metallic implants in the skull, cranial deformities, or absence of cranial vault.
• Cardiac arrhythmias or atrioventricular block.
• Congenital abnormalities, including congenital heart disease or pulmonary malformations.
• History of seizures.
• Use of medications that significantly interfere with autonomic nervous system function, such as antiarrhythmic drugs or insulin.
• Inability to perform the proposed physical and virtual reality-based exercises.
• Inability to tolerate neuromodulation or electrostimulation procedures.
• Non-attendance at baseline or post-intervention assessment sessions, or absence in three or more intervention sessions.
• Incomplete completion of study procedures or withdrawal for any reason.

Participants, care providers, and outcome assessors will be blinded to group allocation. The researcher responsible for delivering TMS will not be blinded due to the nature of the intervention. Data analysis will be conducted using coded group allocation.