The Neural Underpinnings and the Impact of Wearable Sensory Stimulation During Split-belt Treadmill Adaptation in People With Multiple Sclerosis
Phase: NA
Conditions: Multiple Sclerosis
Sponsor: Colorado State University

Document: 
Study Protocol and Statistical Analysis Plan 
 
Official Study Title:  
The Neural Mechanisms of Split-Belt Treadmill Adaptation in People with Multiple Sclerosis 
NCT05878873 
 
Document Date: 
07/25/2023 

 
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Version date: 07/25/2023 
Principal Investigator: Brett Fling 
Study Title: Neural Underpinnings and Sensory Feedback Augmentation During Split-belt Treadmill 
Training in People with Multiple Sclerosis 
I. 
Aim and Hypotheses 
A. Study Objectives, Aim and Hypotheses  
1. Brief Intro: 93.7% of people with multiple sclerosis (PwMS) have impaired mobility and 
commonly report walking dysfunction as the largest factor affecting their quality of life and 
have a higher risk of falls and musculoskeletal injury. Split-belt treadmill training, where the 
speed of each leg is controlled independently, alters each leg’s spatial and temporal 
stepping pattern, and improves gait symmetry in people with Parkinson’s disease and 
stroke. Our recent work is the first to demonstrate strong evidence for similar gait 
improvements in PwMS. In addition, sensory stimulation produces improved gait 
performance and motor coordination within these populations. However, there is limited 
research investigating the neural mechanisms underlying these gait adaptations and 
whether sensory augmentation can elicit enhanced adaptation during split-belt treadmill 
training. 
2. Study Objectives: The objective of this study is to investigate the neural mechanisms of gait 
adaptation and the effects of transcutaneous electrical nerve stimulation (TENS) on 
adaptability during split-belt treadmill training in PwMS. Functional near-infrared 
spectroscopy (fNIRS) will be used during a split-belt treadmill training paradigm to assess 
cortical activation during gait adaptation. Additionally, the effect of split-belt treadmill 
training coupled with TENS on gait adaptability in PwMS will be tested with active and 
inactive TENS during split-belt treadmill sessions. Cortical activation and the effect of TENS 
on gait adaptability will be compared between neurotypical adults and PwMS to assess 
differences that can be attributed to multiple sclerosis. Specifically, we hypothesize that the 
sensorimotor integration areas of the cortex including the supplementary motor areas 
(SMA), premotor cortex (PMC), somatosensory cortex (S1), and posterior parietal cortex 
(PPC) will have heightened activity during split-belt treadmill training, compared to when 
the belts are moving the same speed. Additionally, we hypothesize that the application of 
TENS during split-belt treadmill training will significantly increase gait symmetry 
improvements when compared to split-belt treadmill training alone and will also result in 
improved adaptation retention. 
 
B. Background and Significance  
1. Study Significance: Studies have consistently demonstrated that split-belt treadmill training 
can acutely improve gait symmetry, however there is minimal application of this paradigm 
toward enhancing or prolonging these improvements. The impact of this study is not only 
revealing the cortical mechanisms of sensorimotor adaptation and improving gait symmetry, 
but a necessary step towards translating these improvements out of the lab into a real-
world environment thereby persistently improving mobility for not only PwMS but the tens 
of millions of individuals who experience gait dysfunction. 

 
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2. Our recent studies showed robust gait symmetry improvements in PwMS following split-belt 
treadmill training and additionally has demonstrated that TENS improves positional 
awareness and motor learning during a stepping task. The combination of these findings 
leads us to want to investigate the neural mechanisms of split-belt treadmill training and if 
TENS can enhance to effects of split-belt treadmill training. 
3. Literature Review: Many studies have demonstrated that split-belt treadmill training can 
alter gait symmetry in people with stroke, Parkinson’s disease, and most recently in PwMS. 
This split-belt treadmill training modality has over 20 years of investigation, yet the neural 
mechanisms of sensorimotor adaptation remain poorly understood, especially at the cortical 
level. Along with our preliminary work with TENS, Ng and colleagues found that TENS 
significantly increased gait speed and timed-up-and-go performance post-stroke and 
additionally, Almuklass et al. found that TENS generated immediate improvements of gait, 
balance, and motor skills for PwMS. 
4. Background: We specifically hypothesize that the sensorimotor integration areas of the 
cortex (including SMA, PMC, S1, and PPC) will have heightened activity during split-belt 
treadmill training due to findings from studies utilizing positron emission tomography (PET) 
during split-belt treadmill walking, findings from studies measuring brain activity during dual 
task walking, and the necessity of these regions to process changing spatial information, 
such as one leg moving twice as fast during split-belt treadmill training. Additionally, 
through studies suggesting that TENS improves sensory signaling and gait performance we 
predict that this modality will also improve gait adaptability during split-belt treadmill 
training.  
5. This is the first study to measure real-time cortical activation during split-belt treadmill 
training in any population. This proposal not only addresses the lack of research regarding 
the cortical mechanisms that accompany split-belt treadmill training but also evaluates a 
method to enhance adaptation by coupling the training with sensory augmentation via 
TENS. 
II. 
Research Plan and Design  
A. Study Type and Design: This is a crossover study with both PwMS and age and sex-matched 
neurotypical controls. 30 PwMS and 20 neurotypical controls be randomized into two groups 
and will participate in two split-belt treadmill training sessions while outfitted with cortical fNIRS 
and TENS devices on their bilateral tibialis anterior and rectus femoris. The first group will 
complete split-belt treadmill training with TENS OFF during the first visit and TENS ON during 
the second visit. The second group will complete split-belt treadmill training with TENS ON 
during the first visit and TENS OFF during the second visit to control for a learning effect. In 
between visits there will be a 4-week washout period. Participants will receive TENS only during 
the 10-minute split-belt adaptation period of the paradigm. During all other periods the TENS 
will be OFF.  
B. Sample size, statistical methods, and power calculation 
1. 
Total number of subjects who will be approached (including screen fails, controls and 
subject withdrawals) to reach enrollment numbers for the lifetime of the study for this 
investigator’s site: 60 
2. 
Total number of subjects to be enrolled at this site: 50 

 
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3. 
Brief Description/justification for the proposed sample size in lay terms: From our 
preliminary data we computed a large effect size of 1.01 for change in brain activity in the 
SMA from baseline walking to split-belt treadmill training. According to our power analysis, 
to maintain an alpha of 0.05 and a power of 0.8, at least 10 participants per group are 
needed to detect significant changes in brain activity.  
4. 
Simple randomization will occur (TENS first or TENS second) for both groups (PwMS and 
neurotypical controls). 
5. 
No masking will occur in this study. 
 
C. Subject Criteria (See Vulnerable Populations appendix 1, if applicable): PwMS and 
neurotypical controls from ages 18-86. All races and genders are included. 
1. Inclusion criteria:  
• 
A diagnosis of relapsing remitting multiple sclerosis OR a neurotypical adult (ages 
18-86) 
• 
Not experiencing an active relapse 
• 
Able to stand and walk without an assistive device 
• 
Able to walk for three tenths of a mile without stopping to rest 
• 
MS participants must have a neurologist confirmed diagnosis of relapsing-remitting 
MS. 
• 
MS participants must be independently ambulatory in the community, expanded 
disability status scale (EDSS) score < 5 (clinical measure of MS severity).  
• 
Participants must be 18-85 years of age at the time of participation in the study.  
• 
Participants must be able to stand/walk on a firm surface for least 30 minutes.  
• 
Participants must be able to stand from a seated position on their own volition. 
2. Exclusion criteria:  
• 
Unable to walk for 500 meters without assistance 
• 
Musculoskeletal injury in past 6 months 
• 
Lower extremity surgery in past 6 months 
• 
Unable to abstain from medications that impair balance 
• 
Currently pregnant 
• 
History of traumatic brain injury 
• 
History of vestibular disease 
• 
History of any other balance impairment unrelated to multiple sclerosis 
• 
MS participants must not have primary progressive MS  
• 
MS participants must not have on-going MS relapse  
• 
MS participants must not have other neurologic conditions besides MS, and 
concurrent conditions affecting ability to comply with the study procedures including 
musculoskeletal injury  
• 
MS participants must not have excessive fatigue to the extent that participants can 
not complete all testing required.  
• 
 Participants must not have significant vision impairment 
3. Withdrawal/Termination criteria: Participants will be withdrawn if they are upon baseline 
walking assessment the researcher determines that they do not have enough mobility to 
complete the split-belt treadmill training paradigm. 

 
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4. Participants are allowed to be concurrently enrolled in another research study, unless this 
study causes them to fall within the exclusion criteria. 
 
III. 
Subject Participation 
 
A. Recruitment:  
1. Subjects will be recruited from the local community. We typically recruit from the North 
Central Colorado Urban Area. This 13-county Urban Area comprises the four contiguous 
Metropolitan Statistical Areas of north central Colorado including the Denver-Aurora-
Broomfield, Fort Collins-Loveland, Boulder and Greeley. 
2. Multiple sclerosis affects all races, all genders, and all ages, but is most prevalent in women 
of Northern European descent. This study will recruit individuals with multiple sclerosis, with 
no restrictions for race or gender. We therefore expect the study population to reflect the 
general demographics of multiple sclerosis. Our lab group is well versed in the nuances of 
minority population enrollment to facilitate equity recruitment and in clinical trial 
participation. Our inclusion criteria of 18-86 years were chosen for comparison of clinical 
and biological differences between individuals with multiple sclerosis and neurotypical 
controls across a variety of age groups. These age groups will allow us to detect meaningful 
differences between individuals with multiple sclerosis and neurotypical controls across the 
lifespan. Additionally, we will be poised to identify changes in function as a result of multiple 
sclerosis and will be able to avoid age as a confounding factor due to age and sex matching. 
Our lab group has previously studied multiple sclerosis among this age range for 10+ years 
and have the necessary accommodations (e.g., bodyweight harness, handrails, wheelchairs) 
for all levels of disability status. 
3. Over 100,000 people within the Colorado-Wyoming region are afflicted with MS and 
participant recruitment will be determined in consultation with Dr. Augusto Miravalle from 
the Department of Neurology / Rocky Mountain MS Center at the University of Colorado 
where over 8,000 patient visits are conducted annually. Participants will be recruited from 
the UC Health Regional Neuroscience Center at Poudre Valley Hospital and the Multiple 
Sclerosis Center of the Rockies in Fort Collins as well as the Local National MS Society and 
other local MS and general Neurology clinics. Potential participants will be identified through 
word of mouth, clinician support, or flyers. Interested participants will be contacted via 
email or telephone where they will be informed in greater detail what the study involves. 
Following an explanation of the study potential participants will be asked if this particular 
study is of interest to them and if so a screening for eligibility will be scheduled.  
4. In Kuali, we have attached a copy of a flyer that will be distributed via email to people at 
the Brain Health Center of the Rockies, the Center for Healthy Aging, and the Colorado-
Wyoming chapter of the National Multiple Sclerosis Society. 
5. An email template used to introduce participants to the research study is also included and 
attached in the recruitment folder in Kuali. 
 
B. Screening Procedures or Interview/questionnaire: Demographic information, health 
history (screening), and general physical activity questions will be done prior to beginning the 
testing session and is recorded REDCap via private survey's sent to the participant’s private 
email. All questions asked to participants are attached in the attachment section. They are 

 
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meant to provide the research staff an idea as to whether or not the participant would qualify 
for the study. These various forms give a better picture of the participants unique symptoms 
and their mobility status to be sure that participation is safe for them as well as make 
associations between collected data and reported status. An important confounder to consider 
when walking is cognitive status. The BDI and MOCA will provide additional information about 
cognitive and emotional status that will allow us to have a better full picture of the participant 
when analyzing their data. Only trained graduate students will be allowed to perform screening 
and determine eligibility. Additionally, if the graduate student is unsure due of eligibility due to 
symptom characteristics, the PI, Brett Fling, will be contacted to confirm. The Pi, Brett Fling will 
review inclusion and exclusion criteria of participants weekly to confirm eligibility. 
 
C. Informed consent process and timing of obtaining of consent  
1 
Trained graduate students will be the only personnel allowed to deliver the informed 
consent. Andrew Hagen will be the sole consenter for this study. 
2 
The consent process will include a written and signed document along with a trained 
graduate student verbally describing the consent form. The participant will initial and date 
each page of the consent form, along with signing the last page. The trained graduate 
student will describe the study in lay terms in person and the participant will be allowed to 
ask questions. The consent process will be before any of the study procedures to confirm 
the participant is completely informed and consented to all portions of the study before 
enrolled. 
3 
Trained graduate students and the principal investigator will determine whether the subject 
is able to give consent. People with cognitive impairments and minors will be excluded from 
this study due failing to meet the inclusion/exclusion criteria.  
 
D.   Specific procedures and techniques used throughout the study  
 
1. Laboratory tests 
• 
Gait Adaptation. This will be assessed using inertial sensors and 3-D motion capture. 
Both use noninvasive markers that are placed on the lower limbs of the participants. Our 
outcome measures assessing gait adaptation will be step length asymmetry (SLA). Step 
length is quantified by the anterior-posterior distance between heel markers at leading 
limb heel strike. Step length asymmetry will be calculated by subtracting the step length 
of the less affected limb from the step length of the more affected limb for each 
consecutive step and normalized to participant stride lengths and will be assessed 
continuously throughout each gait trial. We will use step length asymmetry to identify 1) 
the adaptation rate during the split-belt adaptation period to assess speed of adaptation 
between conditions and groups, and 2) adaptation savings, or the difference in 
adaptation rate between visit 2 and visit 1, to quantify adaptation retention between 
conditions and groups.  

 
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• 
Cortical Activation via FNIRS. All fNIRS data will be acquired using a mobile 
NIRSport2 device (NIRx Medical Technologies, nirx.net), which will be secured to 
participants’ backs using backpack-like straps and buckles. This head probe was 
designed to measure regions of interest (ROI) that were established a priori. The Juelich 
atlas within the fNIRS Optodes Location Decider (fOLD) toolbox in MATLAB was used to 
design our optode montage which identifies channel locations above superficial 
components of the frontal, motor, and sensory regions. Our fNIRS head probe will 
include 16 sources and 15 detectors which will create 48 channels over the designated 
ROIs (Figure 1). Additionally, eight short-separator detectors will be used to measure 
scalp perfusion. All fNIRS data will be acquired and streamed to a laptop using Aurora 
software v.2021.9 (NIRx Medical Technologies, nirx.net/aurora). Raw fNIRS data will be 
processed using a commercial software tool, Satori v.1.8 by Brain Innovation (NIRx 
Medical Technologies, nirx.net/satori) to perform pre-processing steps, such as motion 
artifact removal, physiological noise detection, and channel selection and removal. 
Following, the raw light intensity data will be converted to optical density values and 
then converted to oxygenated hemoglobin (HbO), deoxygenated hemoglobin and total 
hemoglobin values using the Modified Beer-Lambert Law automatically through Satori 
software. The HbO beta values calculated from general linear models will be used as a 
proxy for cortical activity, and we will measure changes in HbO beta between each 
protocol period described in Figure 2B.  
 
2. Study Procedures:  
• 
Split-belt Treadmill Training Paradigm. The participants will be outfitted with 6 
APDM Opal inertial sensors (APDM Inc, Portland, OR) as well as 16 retroreflective 
markers for collection of three-dimensional motion capture data during a 10-minute 
split-belt treadmill training paradigm (Figure 2). For the overground walking portion, 
participants will be asked to walk back and forth down a 30m hallway for 2 minutes 
while APDM Mobility Lab is collecting inertial sensor gait cycle parameters. For the 
treadmill portion, participants will be asked to walk on a custom-built instrumented, 
Figure 1. Optode montage (16x15) created 
using Brodmann atlas within fOLD to allow 
measurement of cortical hemodynamics for 
premotor, sensorimotor, and posterior 
parietal regions. Each red circle indicates a 
source optode, each blue circle represents 
a detector optode, and each purple 
connecting line represents a unique 
channel. 

 
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split-belt treadmill with two Bertec force platforms (Model 4060-10, Bertec Corp, 
Columbus, OH) consisting of two separate belts, each with its own motor that 
permits the speed of each belt to be controlled independently and collects ground 
reaction forces at 1000 HZ. The speed at which a participant is waking will be 
individualized to their overground preferred walk speed and fast walk speed. 
Specifically, during the tied-belt walking period, the belts will be set to the 
overground preferred walking speed. During split-belt treadmill adaptation period, 
the “fast belt” speed will be equal to the participant’s fast walk speed while the “slow 
belt” speed will be one half of the participants fast walk speed (2:1 ratio). Prior 
research has determined that the “fast belt” speed should be set for the more 
affected leg to improve gait asymmetry. Following the adaptation period, a 10-
minute deadaptation period with the belts tied at the preferred speed will occur to 
monitor gait symmetry changes during deadaptation between groups.  
• 
TENS Protocol. 30 PwMS and 30 age- and sex- matched controls will be 
randomized into two groups and complete two split-belt treadmill training sessions 
identical to Aim 1, while outfitted with cortical fNIRS and TENS devices on their 
bilateral tibialis anterior and rectus femoris. The first group will complete split-belt 
treadmill training with TENS OFF during the first visit (these are the participants in 
Aim 1) and TENS ON during the second visit. The second group will complete split-
belt treadmill training with TENS ON during the first visit and TENS OFF during the 
second visit to control for a learning effect. In between visits there will be a 4-week 
washout period (Figure 3). Participants will receive TENS only during the 10-minute 
split-belt adaptation period of the paradigm. During all other periods the TENS will be 
OFF.  We will use an FDA-approved clinical TENS device, the LG-TECELITE Therapy 
System (LGMedSupply, Cherry Hill, NJ). Stimulation will consist of bursting biphasic 
pulses delivered with electrode pairs (2 in. × 4 in. pads) placed on the skin over the 
Figure 2. (Top) Time course for the split- belt treadmill training paradigm depicting the stages of baseline, 
adaptation, and deadaptation (Top) fNIRS block design collection protocol. Following a 1-minute baseline 
rest period, fNIRS will be collected during tied-belt walking, split-belt treadmill training, and deadaptation 
utilizing an average of two 30-second blocks for each period of the paradigm to assess differences in 
activation between each period. 

 
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stimulated muscles. The stimulus frequency will be set at 5 Hz with a seven pulses of 
0.15 ms based on recent evidence suggesting that bursting TENS is most effective 
for improving walking performance and limits nerve accommodation to the 
stimulation. The amplitude of the TENS stimulation will be individualized for each 
participant in accordance with their specific motor threshold. The TENS amplitude will 
be slowly increased at 1 mA increments on each individual muscle until non-
voluntary muscle contractions can be seen or felt by the investigator, then the 
amplitude will be set to 2 mA below the motor threshold for each muscle and limb 
during the split-belt adaptation period. Additionally, we are choosing to outfit TENS 
on the tibialis anterior and rectus femoris due to previous studies successfully 
improving gait performance in PwMS using these stimulation locations (Figure 3). 
• 
Clinical Assesments 
During the first visit participants will complete clinical testing to gain a more holistic 
view of their symtpoms. The first test a vibration sensory threshold assessment on 
the left and right hallux. This threshold testing involves the use of  Rydel-Seiffer 
vibrating tuning fork. This tuning forks provides a visual measurement of vibration, 
allowing for a quantifiable measurement of vibration perception across participants. 
It has also been shown in recent work that lower limb sensory perception is 
associated with split-belt treadmill adaptation. Additionally, we will include the 
modified Clinical Test of Sensory Interaction for Balance (mCTSIB) using the BTrackS 
system. This is a very commonly used clinical assessment for balance that is 
noninvasive and easy to implement. This test can distinguish the function of different 
domains of balance individually, including vision, proprioception, and vestibular 
function. An assessment of each balance domain will additionally allow us to better 
quantify the factors that contribute to successful adaptation on the split-belt 
treadmill. Both of these assessments will be performed by a postdoctoral fellow in 
our lab, Dr. Kristin Johnson, who is a board-certified doctor of physical therapy (DPT) 
and will take around five minutes in total. 
 
• 
Photo/Video Use 
Figure 3. (Left) Study design schematic including randomization and TENS condition at each visit. (Right) 
Example of TENS electrode placement on the bilateral tibialis anterior and rectus femoris. 
 
 
 
Randomization 
TENS Second Group 
TENS First Group 
Four-week 
period 
between 
visits 
TENS OFF 
TENS ON 
TENS ON 
TENS OFF 
Visit 1 
Visit 2 

 
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Additonally, photos or videos may be taken for gait analysis purposes, and 
qualitative assesment of gait adaptation. Participants may choose to op in or opt out 
of this via the informed consent form. 
3. All Procedures are for research purposes only and are not a part of standard therapy. 
4. No biospecimens are used in this study. 
 
E. Costs to Subjects: The only cost to participants is transportation to our lab and their time 
(approximately 4-6 hours over two sessions). 
F. Payment, including a prorated plan for payment: Participants will be compensated via 
cash. Participants will receive a total of $100.00 for completing the study. If a participant only 
finishes the first day of the study that participant will receive $50.00. Upon completion of the 
second day that participant will receive an additional $50.00. If a participant drops out or 
withdrawals from the study partway through a session that participant will still be compensated 
$50.00. 
G. Research-related injury: The Colorado Governmental Immunity Act determines and will limit 
Colorado State University's legal responsibility if an injury happens because of this study. 
Claims against the University must be filed within 180 days of the injury. 
H. Risk/benefit assessment: Greater than minimal risk 
1. Physical risk: Yes.  
i. Musculoskeletal injury. There is a low risk of joint, tendon, or muscle pain, inflammation, 
or swelling during or after a testing session of your gait, and balance. This risk is 
reduced through the use of well-trained assistants and also by the mild nature of the 
gait and balance testing. 
ii. Falls. The walking tasks may cause participants to lose their balance and fall. However, 
during the overground walking trials the research team is trained to walk alongside 
participants at all times; if a participant happens to lose their balance, research staff will 
be there to assist and prevent a fall. For the treadmill walking trials, participants will be 
wearing a harness attached to a secure beam to ensure safety and security while 
walking. All safety measures will be taken to ensure a secure and comfortable 
environment. Participants will be allowed to take a break from walking tasks whenever 
necessary. It is not likely that a participant will fall. 
iii. Headache. Although rare, a potential side effect of our brain measurement is headache 
due to pressure on the scalp. However, we have many adjustments available in our cap 
to make the participant as comfortable as possible and mitigate this risk. 
iv. Skin Irritation. The electrical impulses that a TENS unit produces may cause a buzzing, 
tingling, or prickling sensation, which some people may find uncomfortable. Some 
people may be allergic to the adhesive pads. In rare cases, patients have reported burns 
at the sites where the electrodes are placed. However, we use a very mild level of 
stimulation which greatly reduces this discomfort. A TENS unit consists of a battery-
powered device that delivers electrical impulses through electrodes (or sticky pads) 
placed on the surface of your skin. Overall, for the vast majority of people, TENS is 
believed to be safe and well-tolerated with little to no side effects. However, 

 
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manufacturers of TENS units universally warn individuals with pacemakers, epilepsy, or 
are pregnant that these conditions are contraindications to use as they can lead to 
potential complications. We are only placing TENS on the legs, so these warnings will not 
be relevant to this study. 
2. Psychological risk: Yes.  
i. Breach of confidentiality: Although robust protocols and procedures are in place to keep 
your Protected Health Information private and secure, there is a chance of a breach of 
confidentiality. 
ii. Emotional distress. Some of the questions asked may be personal or embarrassing. 
Participants may refuse to answer any potentially uncomfortable question. Also, 
participants may learn information about their balance and walking that could be 
potentially upsetting. 
3. Social risk: No  
4. Economic risk: No  
5. Potential benefit of participating in the study  
a. None 
b. This study will further elucidate differences in neurological activity and the potential 
of TENS to be a rehabilitation strategy for PwMS. 
c. The results of this study will help determine the connections between walking 
asymmetry and brain activity, and the potential of TENS which will be helpful when 
working with diseased populations. 
I. Location where study will be performed: All portions of the study will be performed in the 
Sensorimotor Neuroimaging Laboratory at Colorado State University. 
J. Collaboration (with another institution, if applicable): N/A 
K.  Single IRB Review for a Multi-site study (if applicable):  N/A 
L. Community-Based Participatory Research (if applicable): N/A  
M. Personnel who will conduct the study, including: 
1. Primary responsibility for the following activities, for example:  
a. Determining eligibility: Andrew Hagen 
b. Obtaining informed consent: Andrew Hagen 
c. Maintaining participant's research records: Andrew Hagen 
d. Drawing / collecting laboratory specimens: Andrew Hagen 
e. Performing / conducting tests, procedures, interventions, questionnaires: Andrew 
Hagen 
 
N. Assessment of Subject Safety and Development of a Data and Safety Monitoring Plan 
 
1. Please note that any study proposal with more than minimal risk must include a data and 
safety monitoring plan.  Elements of the plan should include the following: 
a. Persons/groups who will review the data (study team; independent safety monitor, 
data monitoring committee or formal DSMB). This study is a registered clinical trial 
on clinicaltrials.gov (NCT05878873) where Dr. Brett Fling is the lead investigator and 
will be responsible for trial monitoring. Confidentiality will be strictly maintained to 
minimize psychological and economical risks. Participant identities will be kept 
confidential via participant identification numbers (i.e. SBNT_000). The computer 

 
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that will store the project database will be protected by current network security. An 
institutional computer account and password will be required to access the computer 
storing the recruitment database and the REDCap database will be password 
protected, and only project personnel approved by the PI will be given access. Only 
those individuals listed on the IRB consent form and within the Kuali IRB protocol will 
have access to the data. 
b. Data/events that will be reviewed: Objective, de-identified data will be reviewed in a 
rolling fashion throughout the course of this project to allow for dissemination of 
preliminary results and for the use of preparing future grants based upon results. 
Any adverse events that occur will be documented, reported and reviewed 
immediately. 
c. Frequency of review:  Data will be reviewed monthly by our team.  
d. Types of analyses to be performed: Analyses include objective outcomes from the 
split belt treadmill training protocol. This includes gait kinematics and kinetics, as 
well as cortical activation as assessed by fNIRS data.  
e. Safety-related triggers that would cause the PI to alter the study/remove a 
participant: If an individual was unable to safely ambulate either on their own within 
the hallway, or on our treadmill in a safe and efficacious manner, participation would 
be halted immediately. Triggers that would identify an individual as a potential risk 
include repeated stumbling, tripping, or falls where our body weight support harness 
catches the individual.  
2. Describe how adverse events (anticipated and unanticipated) and unanticipated problems 
will be ascertained and handled.  Explain exactly which type of problems will be considered 
serious and reported to the IRB. The reporting timeframe should also be detailed. The 
primary adverse event that could result from this protocol is an unintended fall by a 
participant. To mitigate any risks that would occur as a result of a fall we implement a 
number of safety measures including the use of a “gait belt” while walking overground and a 
body weight support harness while walking on our treadmill. These are standard of care 
practices and the PI of this project has conducted similar mobility-based research for nearly 
two decades and has yet to report an adverse event as a result of an accidental fall. If an 
individual were to fall during this project, we would immediately contact the medical director 
of the HPCRL, Dr. David Thompson for acute assessment. Following this, we would report to 
the IRB within 2 business days, as we believe any unintentional fall is an adverse event and 
requires reporting. 
3. Explain exactly what will happen if a patient experiences an adverse event or other problem 
(for example, will discontinue study participation). As describe above, immediate attention 
would be provided by our in-house physician and medical director, Dr. David Thomson and 
further steps will be conducted under his advisement. If an individual were to experience an 
accidental fall, we would discontinue study participation at that point.  
 
IV. 
Data Collection and Protection 
A. Data Management and Security:  
1. 
All data collected, including identifiable information or personal health information will be 
stored either electronically on secured Colorado State University server or through a 
REDCap database with access restricted to study personnel. Any data not in electronic form 

 
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will be stored in a locked file cabinet with access limited to study personnel. The 
Sensorimotor Neuroimaging Laboratory is located in Moby room 154 within the Department 
of Health and Exercise Science. All hard copy data will be stored in the Sensorimotor 
Neuroimaging Laboratory, with access being restricted to authorized key holders and key 
study personnel. 
2. 
Brett Fling, Kristin Johnson, and Andrew Hagen will have access to the study data. 
3. 
All consent information will be stored electronically on a secure server through Colorado 
State University accessed only by our designated research team. Information collected 
electronically will be stored on a secure Colorado State University network server to prevent 
any breaches in confidentiality. Additionally, all questionnaires, demographics, and 
screening information will be stored in a REDCap database controlled by CCTSI and only 
approved study personnel will have access this database. Data we stored on the R:/Drive 
which is a secure server or on REDCap. Data will be kept for a minimum of 3 years after the 
completion of the study by Dr. Brett Fling. 
4. 
People will be deidentified and all information will be used with a subject code (e.g., 
SBNT_000, with the key to this code available only as a physical copy stored in of locked 
lab.   
5. 
Andrew Hagen and Brett Fling will have access to and maintain the key to the code. 
6. 
Data will only be linked to a participant via a participant code. 
7. 
All data collected, including identifiable information or personal health information will be 
stored either electronically on secured Colorado State University server or through a 
REDCap database with access restricted to study personnel. Any data not in electronic form 
will be stored in a locked file cabinet with access limited to study personnel. The 
Sensorimotor Neuroimaging Laboratory is located in Moby room 154 within the Department 
of Health and Exercise Science. All hard copy data will be stored in the Sensorimotor 
Neuroimaging Laboratory, with access being restricted to authorized key holders and key 
study personnel. 
8. 
No mobile devices will be used for collection or storage of data. 
9. 
N/A. No identifiable data will be sent outside of CSU. 
 
B. Sample / Specimen Collection: N/A 
C. Procedures to protect subject confidentiality: Individual participant data will be de-
identified with given subject numbers, a master list will be stored separately on the secure 
server. Access to the data will be limited to study personnel. Subject ID example (SBNT_001). 
If we are collecting data on multiple people during a single day, we will leave ample time 
between sessions so that participants will not run into each other. All consent information will 
be stored electronically on a secure server through Colorado State University accessed only by 
our designated research team. Information collected electronically will be stored on a secure 
Colorado State University network server to prevent any breaches in confidentiality. 
Additionally, all questionnaires, demographics, and screening information will be stored in a 
REDCap database controlled by CCTSI and only approved study personnel will have access this 
database. 
 

 
INSTITUTIONAL REVIEW BOARD NON-EXEMPT PROTOCOL TEMPLATE 
 
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V.Reporting 
A. Study results to participants: Upon completion of the study, all enrolled participants will 
receive a page summarizing group results in lay-terms. This will not mention any identifiable 
data and only group results will be present. This page will also include the meaning of the 
results, the clinical application, and the future directions of our work.  
B. Dissemination Plan: Brett Fling will ensure that the clinical trial registered with this study has 
up to date information and that results are submitted to ClinicalTrials.gov as outlined in the 
policy and according to the specific timelines stated in the policy. The results of this study will 
be created into at least three manuscripts and submitted to for publication in peer reviewed 
journals. Additionally, both Brett Fling and Andrew Hagen will present the results of the study in 
the form of workshops, podium presentations, and poster presentations at local, regional, 
national, and international conferences within 2 years from the start of the study. 
C. Statistical Analysis Plan: In this crossover design, PwMS and healthy control participants will 
be pseudo-randomized to begin in either the TENS First or TENS Second condition. 
Randomization will be performed using a random number generator and will be 
counterbalanced across age, sex, and fast limb to ensure group balance. A predefined list of 
participant numbers with corresponding randomized assignments will be generated prior to 
enrollment. Linear mixed-effects models will be used to identify differences in adaptation and 
fNIRS outcomes, with group (PwMS vs. healthy controls), visit (Visit 1 vs. Visit 2), and TENS 
condition (TENS ON vs. TENS OFF), along with their interactions, as fixed effects, and 
participant number as a random effect. Assumptions of linearity, normality of residuals, and 
homoscedasticity will be tested for each model. Following model creation, a repeated measures 
ANOVA will be performed for each adaptation outcome (adaptation rate and adaptation savings) 
and for cortical activation (HbO beta). Type III ANOVAs with Satterthwaite’s method for 
degrees of freedom estimation will be used for all analyses. Post–hoc comparisons will be 
conducted using estimated marginal means (EMM) to further investigate significant main effects 
and interactions. P–values will be corrected using the Benjamini–Hochberg false discovery rate 
within each outcome and group when applicable. Cohen's d will be calculated to quantify effect 
sizes. Separate regression models will be conducted to assess the influence of demographic 
covariates on primary outcome models. All statistical analyses will be conducted using the lme4 
and emmeans packages within R and were two-tailed with an alpha threshold set at 0.05.