THE EFFECTS OF AEROBİC EXERCİSE ON NEUROVASCULAR UNİT AND CLİNİCAL PROGRESSİON MARKERS İN INDİVİDUALS WİTH MULTİPLE SCLEROSİS
Phase: NA
Conditions: Multiple Sclerosis
Sponsor: Lokman Hekim University

CLINICAL STUDY RESULTS DOCUMENT 
 
Official Study Title: 
Effects of Aerobic Exercise on Clinical Progression Outcomes and Neurovascular Unit–
related Biomarkers in Individuals with Multiple Sclerosis: A Randomized Controlled 
Trial 
 
Clinical Trial Number: 
- 
 
Document Type: 
Clinical Trial Results / Clinical Study Report 
 
Document Date: 
May 2025 
Study Start: February 1, 2025  
Study Completion: June 30, 2025   
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Study Design 
This study was designed as a randomized controlled trial to comprehensively investigate 
the effects of aerobic exercise on NVU biomarkers in individuals with MS. The study 
was conducted between February and June 2025 at the MS outpatient clinic of the 
Department of Neurology, Ondokuz Mayıs University Faculty of Medicine. Participants 
were randomly assigned to either a control group or an exercise group. Assessments were 
performed at baseline (pre-intervention) and eight weeks after the intervention (post-
intervention). The study procedure is summarized in the CONSORT flow diagram 
(Figure 1). No modifications or deviations from the original study protocol were made 
after the initiation of the trial. 
 Participants 
Individuals with a definitive diagnosis of MS according to the McDonald criteria who 
presented to the Neurology Clinic of Ondokuz Mayıs University were invited to 
participate in the study. A total of 38 patients who met the inclusion criteria and provided 
written informed consent were allocated into two groups the exercise group (n = 19) and 
the control group (n = 19) using a simple randomization method. Randomization was 
performed by the physician responsible for patient referral using a computerized random 
allocation sequence. Importantly, the referring physician was blinded to the specific 
interventions applied in each study group. Outcome assessors and laboratory personnel 
responsible for biochemical analyses were also blinded to group allocation throughout the 
study. Group allocation was concealed until the completion of baseline assessments. Due 
to the nature of the exercise intervention, participant and therapist blinding was not 
feasible, and therefore neither participants nor therapists were blinded to group 
allocation. 
A total of 38 participants were randomized into the study. During the follow-up period, 
four participants in the control group did not complete the study (one due to an MS 
relapse, one due to non–MS-related health problems, and two due to non-attendance at 

the final assessment). In the exercise group, four participants did not complete the study 
(two due to non-completion of the exercise program and two due to changes in 
pharmacological treatment). Thirty participants completed the intervention and post-
intervention assessments (control group: n = 15; exercise group: n = 15) (Figure 1). 
Inclusion criteria were as follows: a definitive diagnosis of MS according to the 
McDonald criteria; age between 20 and 50 years; a Standardized Mini-Mental Test 
(SMMT) score of ≥24; an EDSS score between 0 and 4.0; absence of any other serious 
medical condition that would preclude exercise participation; and no changes in 
pharmacological treatment within the previous six months. 
Exclusion criteria included: experiencing an MS relapse within the past six months; 
presence of orthopedic or systemic conditions that would limit participation in exercise; 
having a known neuromuscular disorder other than MS; receipt of immunomodulatory 
treatment within the previous six months; presence of cardiopulmonary conditions that 
could contraindicate exercise; and pregnancy or breastfeeding. 
Withdrawal criteria were defined as: failure to attend three consecutive exercise sessions; 
changes in or interruption of ongoing pharmacological treatment; occurrence of an MS 
relapse; or withdrawal from the study due to health-related or personal reasons. 
 
Measures 
Participants were assessed before group allocation (baseline assessment) and at the eighth 
week following completion of the aerobic exercise program (post-intervention 
assessment). All participants with a definitive diagnosis of MS underwent the 
Standardized Mini-Mental Test (SMMT), and their EDSS scores were determined. 
Patients who scored ≥24 on the SMMT, had an EDSS score between 0 and 4.0, and met 
the inclusion criteria underwent baseline assessments to determine clinical progression. 
These assessments included a demographic data form, the 6MWT, the 9HPT, and the 
TUG. In addition, blood samples were collected to evaluate NVU related biomarkers. 

Following completion of the aerobic exercise program, the 6MWT, 9HPT, and TUG 
were re-administered by the researchers, post-intervention blood samples were obtained, 
and final assessments were completed. All clinical evaluations were conducted by expert 
physiotherapists with experience in neurological rehabilitation. To ensure objectivity of 
the measurements, the assessors were blinded to the results of the biochemical analyses. 
Demographic Data Form: 
A demographic data form was developed for this study in accordance with the literature 
to collect information on participants’ demographic characteristics and MS-related 
symptoms (Knowles et al., 2024). The form included data on age, sex, use of assistive 
devices, MS type, date of MS diagnosis, number and timing of relapses, time since the 
most recent relapse, current medications, comorbid chronic diseases, history of surgical 
interventions, presence of fatigue, and exercise habits. 
Expanded Disability Status Scale (EDSS): 
The EDSS is a practical and reliable scale frequently used in clinical settings to evaluate 
disease progression and treatment response in individuals with MS. It was originally 
developed by neurologist John Kurtzke as a 10-step Disability Status Scale. The EDSS is 
widely used by clinicians worldwide to assess functional systems of the central nervous 
system in individuals with MS. Scoring is based on the extent of impairment in MS-
affected body functions, and the total score constitutes the EDSS value, ranging from 0 to 
10 (Demir, 2022). 
Standardized Mini-Mental Test (SMMT): 
The SMMT consists of 11 items grouped into five main domains: orientation, registration 
memory, attention and calculation, recall, and language. Scores between 0–9 indicate 
severe cognitive impairment, 10–19 moderate cognitive impairment, 20–23 mild 
cognitive impairment, and 24–30 normal cognitive function. The validity and reliability 
of the SMMT for the Turkish population were established by Güngen et al. (Güngen et 
al., 2002). 
6-Minute Walk Test (6MWT): 
The 6MWT is a clinical exercise test used for the objective assessment of functional 

exercise capacity and provides an estimate of disease-related effects of MS on walking 
endurance. The test measures the distance walked by the participant over six minutes on a 
flat 30-meter corridor. In healthy individuals, the expected walking distance ranges 
between 400 and 700 meters. Blood pressure, oxygen saturation, heart rate, and perceived 
levels of dyspnea and fatigue using the Borg scale are assessed before and after the test. 
If the participant stops to rest during the test, the stopwatch is not paused. The test is 
terminated in the presence of chest pain, intolerable dyspnea, leg cramps, unsteadiness, 
diaphoresis, cyanosis or pallor, or if oxygen saturation falls below 85% (Savci et al., 
2005). 
9-Hole Peg Test (9HPT): 
The 9HPT is used to assess upper extremity function and fine motor skills in clinical 
populations. The test requires a wooden board with nine holes of standardized diameter 
(0.71 cm), depth (1.3 cm), and spacing (3.2 cm), along with nine cylindrical pegs of 
standardized diameter (0.64 cm) and length (3.2 cm). During the test, the participant is 
timed while placing the pegs into the holes and subsequently removing them. Timing 
begins when the participant first touches a peg and ends when the last peg is released. 
The 9HPT is a valid and reliable test sensitive to clinical changes, and its validity and 
reliability have been established in individuals with MS (Feys et al., 2017). 
Timed Up and Go Test (TUG): 
TUG is widely used in clinical practice and has been shown to be a valid and reliable 
measure for assessing functional mobility and fall risk in individuals with MS. In a study 
conducted by Kalron et al., individuals with MS who experienced recurrent falls (at least 
one fall) had significantly longer TUG times compared with those without falls. 
Moreover, prolonged TUG time in recurrent fallers was identified as an excellent 
predictor of fall risk, supporting its use in fall risk assessment in this population (Kalron 
et al.,2017). 
 Sample Size and Power 
The sample size calculation and power analysis of the study were conducted using the 
G*Power software (version 3.0.10; Universität Düsseldorf, Düsseldorf, Germany). For 

the comparison of changes in the 6MWT, which was defined as the primary outcome 
measure of the study, an analysis of covariance (ANCOVA) was planned in order to 
control for the effect of baseline measurements. For a study design consisting of two 
groups and two measurement time points (pre-test and post-test), assuming an expected 
effect size of f = 0.70, the required sample size was calculated as at least 15 participants 
per group and 30 participants in total, with a Type I error rate (alpha) of 5% and a 
statistical power (1–beta) of 95%. Considering potential drop-outs that might occur 
during the study period, the sample size was increased by approximately 25%, and the 
study was initiated with a total of 38 participants, including 19 participants in each group. 
After completion of the study, a post-hoc power analysis was performed based on the 
primary outcome measure (6MWT values). For an analysis with a Type I error rate of 
5%, a sample size of 30, and an effect size (f) of 2.16, the post-hoc statistical power (1–β) 
was found to exceed 99.99%.  
 Intervention 
The aerobic exercise program was developed in accordance with the existing literature 
with the aim of improving functional capacity, reducing disease progression, and 
promoting favorable changes in NVU-related biomarkers in individuals with MS 
(Learmonth and Givon, 2017). The program designed for individuals with MS was 
scheduled for eight weeks, with two sessions per week. Participants who met the 
inclusion criteria, had SMMT scores ≥24, EDSS scores between 0 and 4.0, and provided 
written informed consent were randomly assigned to one of two groups: the control group 
(C) or the exercise group (E). 
Following group allocation, baseline assessments—including the 6MWT, TUG, and 
9HPT were conducted. After completion of baseline assessments, participants in the 
exercise group received an individualized aerobic exercise program tailored to their 
functional capacities and were supervised for eight weeks, with two sessions per week 
lasting 30–40 minutes each. In addition, a conventional exercise program tailored to 
individual needs was prescribed as a home exercise program. Participants in the control 
group were advised to perform only the conventional exercise program at home, 
according to their individual needs. At the end of the eighth week, both groups underwent 

post-intervention assessments, including the 6MWT, TUG, and 9HPT, and blood samples 
were collected to complete the evaluations. 
Aerobic Exercise Program 
The aerobic exercise program was implemented under the supervision of an expert 
physiotherapist using a treadmill, with two sessions per week over a total period of eight 
weeks. Participants’ functional capacities were determined by estimating peak oxygen 
consumption (VO₂peak) based on walking distance obtained from the 6MWT, heart rate, 
and blood pressure measurements (Ross et al., 2010). During the aerobic exercise 
sessions, heart rate and oxygen saturation were continuously monitored using a pulse 
oximeter, and exercise intensity was adjusted to maintain a perceived exertion level 
between 11 and 14 on the Borg Rating of Perceived Exertion Scale. 
Following completion of each aerobic exercise session, participants were monitored for at 
least five minutes until heart rate, blood pressure, and Borg scale ratings returned to 
baseline values. Session duration was planned in accordance with the literature and 
consisted of three phases: warm-up, training, and cool-down, with a total duration 
ranging from 30 to 40 minutes. The warm-up phase lasted 5 minutes, followed by a 
moderate-intensity training phase lasting 20–30 minutes at 50–75% of VO₂peak, and a 5-
minute cool-down phase. When necessary, the exercise duration was divided into two 
bouts based on participants’ fatigue levels. Progression of exercise intensity (i.e., walking 
speed) was increased gradually by 0–5% per week, based on heart rate responses and 
perceived exertion (Halabchi et al., 2017). 
Conventional Exercise Program 
The conventional exercise program prescribed according to individual needs included 
exercises aimed at strengthening the lower extremities and trunk, balance and gait 
training exercises (progressing from firm to soft surfaces, from eyes open to eyes closed, 
and from wide to narrow base of support), gait training exercises (marching gait, walking 
with reduced base of support, and lateral walking), and coordination exercises (Frenkel 
coordination exercises performed in sitting or supine positions, depending on the 
participant’s condition). Each individualized program consisted of approximately 8–12 

exercises and was recommended to be performed at least two days per week, with each 
session not exceeding one hour in duration (Moeinzadeh et al., 2023). Participants were 
informed about the importance of adhering to the exercise program regularly. 
NfL, GFAP, and VEGF-a Analyses 
Blood samples required for biomarker analyses were collected from all participants after 
a minimum fasting period of 12 hours, between 09:00 and 11:00 a.m. The samples were 
transferred into gel-containing biochemical tubes and centrifuged at 4000 rpm for 5 
minutes. After centrifugation, serum samples were aliquoted into 1.5 mL microtubes and 
stored at −80°C in the Department of Medical Biochemistry, Ondokuz Mayıs University 
Hospital, until the day of analysis. 
On the day of analysis, serum samples were thawed under appropriate conditions and 
diluted at a ratio of 1:2 using the sample dilution buffer provided in the assay kits. 
Analyses were performed using commercially available enzyme-linked immunosorbent 
assay (ELISA) kits based on the sandwich method. Each sample was analyzed in 
duplicate, and the coefficient of variation (CV) between duplicates was calculated. 
Samples with a CV greater than 10% were reanalyzed. As all analyses were conducted in 
a single run, inter-assay variability could not be directly assessed; instead, intra-assay 
repeatability was evaluated. The following ELISA kits were used to measure protein 
levels of the study parameters: Human GFAP (FineTest, Cat. No. EH0410, China), 
Human VEGF-a (FineTest, Cat. No. EH0327, China), and Human NfL (FineTest, Cat. 
No. EH1205, China). 
Ethical Considerations 
Ethical approval for the implementation of this study was obtained from the Scientific 
Research Ethics Committee of Lokman Hekim University (Approval No: 2024313; dated 
31 January 2025), and written permission was granted by Ondokuz Mayıs University 
Faculty of Medicine, where the study was conducted. Prior to participation, all 
individuals were verbally informed about the study procedures, and written informed 
consent was obtained from each participant. 

Outcome Measurements 
Outcome measures of the study were structured into two main categories primary and 
secondary outcomes in order to evaluate the effects of the intervention on clinical 
functional outcomes and NVU-related biomarkers. All measurements were performed 
before randomization (baseline) and at the end of the eight-week intervention period. 
Clinical assessments were conducted by physiotherapists experienced in neurological 
rehabilitation, and the assessors were blinded to the biochemical analysis results. 
The primary outcome of the study was the assessment of clinical progression in 
individuals with MS. Clinical progression was evaluated by measuring functional 
mobility, walking capacity, and upper extremity function using the 6MWT, the 9HPT, 
and the TUG, respectively. In the exercise group, significant reductions were observed in 
dominant and non-dominant hand 9HPT times and TUG time, along with a significant 
increase in 6MWT distance (p < 0.001). No significant changes were observed in these 
parameters in the control group (p > 0.05). Between-group comparisons revealed that 
improvements in the exercise group were significantly greater than those observed in the 
control group (p < 0.001). These findings indicate that the eight-week aerobic exercise 
program had a positive effect on clinical progression markers in individuals with MS. 
Notably, the observed increase in 6MWT distance exceeded the previously reported 
minimal clinically important difference for individuals with multiple sclerosis. 
The secondary outcomes consisted of biomarkers reflecting NVU function. Serum levels 
of NfL, VEGF-a, and GFAP were measured as NVU biomarkers. Measurements were 
performed using the ELISA method on fasting morning blood samples collected from all 
participants. Secondary outcome analyses demonstrated no significant within-group or 
between-group differences in serum NfL, VEGF-a, or GFAP levels (p > 0.05). These 
results suggest that the eight-week aerobic exercise program did not produce a significant 
effect on NVU-related biomarkers. 
In addition, clinical and sociodemographic characteristics, including age, sex, MS type, 
disease duration, relapse history, and exercise habits were recorded using the 
demographic data form. These data confirmed the absence of significant baseline 

differences between groups and allowed for control of potential confounding variables in 
the analyses. 
Data Analyses 
Statistical analyses were performed using IBM SPSS Statistics version 26.0 (SPSS Inc., 
Chicago, IL, USA). The normality of continuous variables was assessed using visual 
(histograms and probability plots) and analytical methods. Descriptive statistics were 
presented as frequencies and percentages for categorical variables, and as medians with 
percentiles or means with standard deviations for continuous variables, as appropriate. 
The Chi-square test was used to compare independent categorical variables. For within-
group (pre-treatment vs. post-treatment) comparisons of continuous variables in 
dependent samples, the Paired Samples t-test was applied for normally distributed data, 
while the Wilcoxon signed-rank test was used for non-normally distributed data. For 
between-group comparisons of continuous variables in independent samples, the 
Independent Samples t-test was used for normally distributed data, and the Mann–
Whitney U test for non-normally distributed data (Barton and Peat, 2014; Field, 2024). 
One-way analysis of covariance (ANCOVA) was used to compare the effectiveness of 
the intervention methods. The assumptions of ANCOVA—normal distribution of the 
dependent variable in both groups, homogeneity of variances between groups, normal 
distribution of residuals, a linear relationship between the covariate and the dependent 
variable, and homogeneity of regression slopes—were tested. When these assumptions 
were not met, Quade’s ANCOVA was applied. For both analyses, post-treatment values 
were assigned as the dependent variable, the grouping variable as the independent 
variable, and pre-treatment values as the covariate. 
Partial eta squared (η²) values obtained from the analyses were used to determine effect 
sizes (Barton and Peat, 2014; Field, 2024). Effect sizes were classified as small (0.01 < η² 
< 0.06), medium (0.06 < η² < 0.14), and large (η² > 0.14) (Cohen, 2013). A p-value of 
less than 0.05 was considered statistically significant for all analyses.