No additive effect of transcranial direct current stimulation on balance exercises for brain activity and clinical outcomes in patients with chronic ankle instability: a randomised controlled trial

Zivar Beyraghi; Roya Khanmohammadi

doi:10.1136/bmjsem-2024-002401

. 2025 Apr 10;11(2):e002401. doi: 10.1136/bmjsem-2024-002401

No additive effect of transcranial direct current stimulation on balance exercises for brain activity and clinical outcomes in patients with chronic ankle instability: a randomised controlled trial

Zivar Beyraghi ¹, Roya Khanmohammadi ^1,^✉

PMCID: PMC11987147 PMID: 40226331

Abstract

Objectives

This study explored whether adding transcranial direct current stimulation (tDCS) to balance exercises enhances preparatory brain activity and clinical outcomes in individuals with chronic ankle instability.

Methods

30 participants were randomised into two groups: balance exercises with real tDCS and balance exercises with sham tDCS. Neurophysiological measures, including late contingent negative variation (CNV) amplitude, peak amplitude and peak time, served as primary outcomes, while biomechanical (anticipatory postural adjustment duration) and clinical (dynamic balance and perceived ankle instability) outcomes were secondary. Both groups completed 12 sessions, each lasting 60 min.

Results

The results revealed no significant group-by-time interaction for late CNV amplitude, CNV peak amplitude, perceived ankle instability scores or dynamic balance, indicating no added benefit of real tDCS over sham. However, both groups demonstrated significant post-treatment improvements in late CNV amplitude (C3, Cz, C4: p≤0.017, η²=0.177–0.276) and CNV peak amplitude at the C3 electrode (p=0.026, η²=0.158), reflecting enhanced preparatory brain activity. Similarly, dynamic balance improved significantly in the anterior, posterior-medial and posterior-lateral directions (p≤0.010, η²=0.204–0.350) and perceived ankle instability scores increased notably, indicating reduced instability (p<0.001, η²=0.391), regardless of the tDCS condition. Furthermore, significant correlations (r=0.381–0.553) were observed between treatment-induced changes in neurophysiological variables and biomechanical and clinical outcomes.

Conclusions

Although tDCS did not show a distinct advantage, the improvements in neurophysiological and clinical outcomes suggest that balance exercises effectively target central mechanisms. Additionally, relationships were found between enhancements in neurophysiological outcomes and other measures, emphasising the pivotal role of central mechanisms in driving these positive effects.

Trial registration number

IRCT20210604051488N1.

Keywords: Ankle, Brain, Exercise rehabilitation, Intervention, Physiotherapy

WHAT IS ALREADY KNOWN ON THIS TOPIC

Chronic ankle instability (CAI) is associated with deficits in both central and peripheral neural mechanisms. Balance exercises are effective in improving motor control and stability, while transcranial direct current stimulation (tDCS) has been explored as a potential adjunct for enhancing neuroplasticity.

WHAT THIS STUDY ADDS

Building on our previous work (Beyraghi et al), this study shifts the focus from biomechanical measures to central neurophysiological mechanisms. By analysing the relationships between neurophysiological outcomes and clinical and biomechanical measures, this study offers deeper insights into the neural mechanisms underlying rehabilitation in individuals with CAI. Our findings indicate that while balance training improves neurophysiological markers, dynamic balance and perceived ankle stability in individuals with CAI, adding tDCS does not confer any additional benefits. Strong correlations between neurophysiological, biomechanical and clinical improvements highlight the central mechanisms’ role in recovery.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

The findings highlight that balance exercises can effectively target central mechanisms and improve outcomes in individuals with CAI, questioning the added value of combining tDCS with such interventions. This emphasises the importance of optimising exercise-based rehabilitation protocols as a primary treatment strategy. Clinically, the study reinforces the value of balance exercises as an evidence-based, stand-alone intervention, guiding practitioners towards effective, non-invasive treatments. From a policy perspective, these findings advocate for prioritising affordable, accessible and scalable exercise-based programmes in rehabilitation services for CAI.

Introduction

Chronic ankle instability (CAI) is a prevalent and debilitating condition characterised by persistent symptoms, including recurrent episodes of the ankle ‘giving way,’ weakness, pain, swelling and significant functional limitations.¹ These issues are commonly linked to insufficient rehabilitation rather than the grade of the initial ligamentous tear caused by a sprain. CAI not only impairs functional performance but also increases the risk of joint degeneration, leading to long-term mobility issues and a substantial decline in quality of life.^{2 3} Despite extensive rehabilitation efforts, the effectiveness of current treatment options for CAI remains inadequate, highlighting the need for more comprehensive and innovative rehabilitation approaches.

The pathophysiology of CAI involves both peripheral and central impairments.⁴ Traditional rehabilitation approaches primarily target peripheral deficits, such as ligament laxity, muscle weakness and impaired joint position sense, but emerging evidence suggests that CAI is also a neurophysiological disorder.⁴ CAI is associated with significant alterations in central nervous system (CNS) function, which contribute to persistent instability and motor deficits.5,7 Brain regions crucial for motor control, such as the supplementary motor area (SMA) and primary motor cortex (M1), show altered activation patterns in individuals with CAI.^{8 9} These neurophysiological changes suggest that CAI involves not just ankle joint dysfunction, but also disruptions in the central mechanisms that govern motor coordination and postural control, increasing the risk of reinjury.

Preparatory brain activity, indicated by the contingent negative variation (CNV), is a critical component of motor control for maintaining dynamic stability during functional tasks, such as transitioning from a double-limb stance to a single-limb stance.^{7 10 11} In healthy individuals, well-coordinated CNV patterns are linked to efficient motor planning and anticipatory postural adjustments (APAs), enabling smooth transitions between movements, such as during gait initiation (GI).^{7 10} However, individuals with CAI exhibit altered CNV patterns, such as reduced late CNV amplitude, a lower CNV peak, an earlier signal peak and impaired postural adjustments, contributing to impaired balance and gait instability.⁷

Given the central and peripheral impairments associated with CAI, there is increasing interest in rehabilitation approaches that target both aspects. Balance exercises are a cornerstone of CAI rehabilitation, primarily addressing peripheral deficits.^{12 13} Traditional balance training does not directly target the central dysfunctions observed in CAI. For example, Burcal et al’s research found that despite improvements in functional tests and balance scores, conventional balance training did not lead to significant changes in brain activity, indicating that traditional rehabilitation approaches may be insufficient in promoting the necessary cortical adaptations.¹⁴ Thus, there is growing recognition that addressing CNS dysfunctions through supplementary interventions may be essential for achieving more comprehensive rehabilitation outcomes.

One promising approach to complement traditional balance exercises is transcranial direct current stimulation (tDCS), a non-invasive neuromodulation technique that modulates cortical excitability through low-intensity electrical currents. Anodal stimulation enhances cortical excitability by inducing a depolarising effect on the neuronal membrane, promoting neuroplasticity.¹⁵

Recent studies suggest that combining tDCS with exercises may improve rehabilitation outcomes in individuals with musculoskeletal disorders, including chronic low back pain^{16 17} and CAI.^{18 19} However, our previous study offers a nuanced perspective, demonstrating that while balance exercises significantly improved APAs, anodal tDCS over the SMA did not offer additional benefits beyond sham stimulation.²⁰ This is despite the SMA’s crucial role in APAs, motor preparation and GI,²¹ as well as evidence linking altered SMA activity to increased perceived ankle instability in individuals with CAI.⁹ A 2018 systematic review of 27 studies underscored the potential of non-invasive brain stimulation techniques, including those targeting the SMA, in enhancing motor preparation by improving reaction times in healthy individuals.²² However, despite promising findings, research on the efficacy of tDCS in musculoskeletal rehabilitation remains limited and inconsistent, with studies yielding mixed results.16,2023 While some studies16,19 have reported notable functional improvements, others^{20 23} have found no significant benefits compared with sham conditions. This variability in outcomes emphasises the need for further rigorous research. Specifically, the impact of tDCS on preparatory brain activity in individuals with CAI remains unexplored. Apart from Bruce et al’s study, most research has not focused on brain-related parameters to examine the link between treatment-induced neurophysiological changes and clinical outcomes.

This study aims to address these gaps by examining the combined effects of tDCS and balance training on preparatory brain activity, dynamic stability and perceived ankle instability in CAI patients. By integrating neurophysiological and functional perspectives, the research seeks to advance our understanding of how neuromodulation can enhance rehabilitation outcomes for CAI, offering a more evidence-based and holistic approach to treatment. Additionally, by addressing both central and peripheral mechanisms of CAI, the study may contribute to the development of more effective and integrative rehabilitation strategies that target the complex nature of the condition. We hypothesised that both groups engaging in balance exercises would exhibit improvements, including increased late CNV amplitude, greater CNV peak amplitude, a closer alignment of CNV peak timing to the response stimulus, reduced APA duration, enhanced ankle stability and better dynamic balance. Furthermore, we anticipated that combining balance exercises with SMA-targeted tDCS would further enhance these effects.

Methods

Trial design

This study used a parallel, randomised, single-blind, sham-controlled design. Participants were evenly assigned to either the real or sham tDCS group, receiving the intervention alongside balance exercises in a 1:1 allocation ratio. Outcome measures were assessed both before and after the intervention. This study was registered as a clinical trial on the Iranian Registry of Clinical Trials (IRCT20210604051488N1) on 28 June 2021.

Participants

30 individuals with CAI participated in the study. Participants were recruited through posters and social media advertisements targeting the university community, sports clubs and federations. A Consolidated Standards of Reporting Trials (CONSORT) flow diagram illustrates participant progression from enrolment to analysis (figure 1).

Participants were included if they met the following criteria: (1) age between 18 and 40 years²⁴; (2) self-reported history of the first acute ankle sprain occurring more than 1 year ago¹; (3) at least two episodes of ‘giving way’ or sensations of ankle joint instability within the past 6 months¹; (4) most recent ankle sprain occurring more than 3 months prior to enrolment¹; (5) Cumberland Ankle Instability Tool (CAIT) score ≤24 (scores) range from 0 to 30, with lower scores indicating greater instability)¹; (6) Foot and Ankle Ability Measure scores below 90% for daily activities and below 80% for sports activities²⁵; (7) no self-reported history of fractures or surgery in the lower extremities; (8) no psychological or neurological disorders; (9) no history of migraines, epilepsy, seizures or head injuries resulting in loss of consciousness; (10) no metallic implants (eg, surgical clips, intracranial electrodes, pacemakers) and (11) no current use of prescription or self-medication.²⁶ Participants were excluded if they missed three or more non-consecutive sessions or two consecutive sessions.

Interventions

The participants were randomly assigned to two groups: (1) the intervention group (balance exercises combined with real tDCS) and (2) the control group (balance exercises combined with sham tDCS). In both groups, tDCS was administered prior to the balance exercises. Each group completed 12 sessions over 4 weeks, with three sessions per week. Each session lasted 60 min, comprising 20 min of tDCS followed by 40 min of balance exercises. All treatments were conducted in a controlled laboratory environment. Participants were allowed to continue their usual daily activities and routine care; however, they were advised to avoid any new rehabilitation programmes or treatments for CAI during the study to ensure that the observed effects were solely attributable to the assigned intervention. Based on self-reports, no participants indicated engaging in additional interventions during the study period.

Balance exercises

The balance exercise programme included a variety of tasks aimed at improving dynamic stability. Activities involved single-leg stance with modifications to visual and proprioceptive inputs, single-leg stance while throwing and kicking a ball, single-leg hopping to stabilisation and hopping to stabilisation with reaching. Details of the specific exercises and progression criteria are provided in table 1.^{12 13 20 27} Participants progressed to more challenging levels only after demonstrating error-free performance at their current level. The required number of error-free repetitions for progression is outlined in table 1. Throughout the exercises, the therapist provided verbal instructions to correct errors and reinforce proper technique. Over time, instructions were gradually reduced to encourage participants to perform the exercises independently. All exercise sessions were conducted individually (in person) under the supervision of a licensed physiotherapist with 5 years of experience in managing patients with ankle instability.

Table 1. The description of balance exercises.

	Difficulty levels	Instructions	Criteria for progression	Errors
Single-limb stance	Eyes open, hard surface, 30 s, 3 Reps Eyes open, hard surface, 60 s, 3 Reps Eyes open, foam surface, 30 s, 3 Reps Eyes open, foam surface, 60 s, 3 Reps Eyes open, foam surface, 90 s, 3 Reps Eyes open, foam surface, 30 s, ball throwing, 3 Reps Eyes open, foam surface, 60 s, ball throwing, 3 Reps Eyes open, foam surface, 90 s, ball throwing, 3 Reps Eyes closed, hard surface, 30 s, arms out, 3 Reps Eyes closed, hard surface, 30 s, arms across, 3 Reps Eyes closed, foam surface, 30 s, arms out, 3 Reps Eyes closed, foam surface, 30 s, arms across, 3 Reps	The participant attempted to maintain balance on the affected limb while facing challenges such as altering the base of support, closing eyes, extending the duration and throwing a ball.^{12 48}	If the participant could complete 3 repetitions without errors at each level of difficulty.	Errors were defined as: Touching the ground with the opposite foot. Excessive trunk motion (more than 30° of lateral flexion). Resting the opposite limb against the stance limb. Lifting the hands from the chest while standing.
Limb stance with ball kicking	Double-limb stance, hard surface, ball Kicking, 3 Reps Single-limb stance, hard surface, ball Kicking, 3 Reps Double-limb stance, mini-trampoline, ball Kicking, 3 Reps Single-limb stance, mini-trampoline, ball kicking, 3 Reps	The participant was instructed to return the ball to the therapist and maintain balance as much as possible after kicking. During this training, the uninjured limb was used for kicking, while the affected limb served as the supporting limb.²⁷	If the participant successfully completed 3 repetitions without any errors at each difficulty level.	Errors included A, B, C and D mentioned earlier.
Single-limb hop to stabilisation	Target at 18 inch, using arms to aid in stabilising, 10 Reps Target at 18 inch, hands on hips while stabilising, 10 Reps Target at 27 inch, using arms to aid in stabilising, 10 Reps Target at 27 inch, hands on hips while stabilising, 10 Reps Target at 36 inch, using arms to aid in stabilising, 10 Reps Target at 36 inch, hands on hips while stabilising, 10 Reps	The participant was instructed to perform 10 hops in four directions: Anterior–posterior Medial–lateral Anteromedial–posterolateral Anterolateral–posteromedial Three target distances (18, 27 or 36 inches) from the starting point were set. The participant hopped to these targets, stabilised their balance on one limb, then hopped back in the opposite direction to the starting position and stabilised again on one limb.⁴⁸	If the participant successfully completed 10 repetitions without any errors at each level of difficulty.	Errors included A, B, C and D mentioned earlier.
Hop to stabilisation and reach	Target at 18 inch, using arms to aid in stabilising, 5 Reps Target at 18 inch, hands on hips while stabilising, 5 Reps Target at 27 inch, using arms to aid in stabilising, 5 Reps Target at 27 inch, hands on hips while stabilising, 5 Reps Target at 36 inch, using arms to aid in stabilising, 5 Reps Target at 36 inch, hands on hips while stabilising, 5 Reps	The participant hopped, stabilised and reached back to the starting position. Then, they returned to the starting position and reached to the target position.⁴⁸	If the participant successfully completed 5 repetitions without errors at each difficulty level.	Errors included A, B, C and D mentioned earlier.

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.Reps, repetitions.

transcranial direct current stimulation

tDCS was administered using a NEUROSTIM2 device (MEDINA TEB GOSTAR, Iran) with two 25 cm² electrodes soaked in sterile saline (0.9% NaCl) to ensure optimal conductivity. The ‘anode’ electrode was positioned 1.8 cm anterior to Cz, corresponding to the SMA according to the international 10–20 EEG electrode placement system. The ‘cathode’ electrode served as the reference and was placed centrally on the forehead, just above the eyebrows.²⁸ For the intervention condition, a current of 1.5 mA was delivered for 20 min, with a 30 s ramp-up (fade-in) at the beginning and a 30 s ramp-down (fade-out) at the end to minimise discomfort. For the sham condition, the same 30 s ramp-up was applied, but the current was discontinued after this initial period, mimicking the sensation of active stimulation without delivering therapeutic effects.²⁰

Both real and sham tDCS sessions were conducted individually in a quiet, temperature-controlled room to ensure participant comfort and consistency. tDCS was administered by a licensed physiotherapist with 2 years of experience. Participants were seated comfortably in an upright position and instructed to remain relaxed throughout the stimulation to optimise the intervention’s effectiveness.

The SMA is a key brain region involved in APAs, motor preparation and GI.²¹ It also plays a significant role in generating the CNV.¹⁰ Supporting these roles, numerous studies have shown that anodal stimulation of the SMA can notably improve reaction times and enhance APAs in healthy individuals.28,31 Additionally, evidence indicates that individuals with CAI exhibit altered SMA activity, with a strong correlation between SMA activation and perceived ankle instability. This altered activity sheds light on the disrupted postural control mechanisms seen in CAI.⁹ Based on these findings, tDCS was used to stimulate this brain region in the present study.

Outcome measures

The independent variables in the study were the group (real vs sham tDCS) and time (pretreatment and post-treatment). The primary dependent variable was late CNV amplitude, as it most accurately represents the key neurophysiological changes associated with the intervention. The secondary dependent outcomes included other neurophysiological measures, such as CNV peak amplitude and CNV peak time, as well as biomechanical outcomes like APA duration and clinical outcomes, including dynamic balance and the severity of perceived ankle instability.

Neurophysiological outcomes

Brain activity was recorded using a 64-channel EEG system with Ag-AgCl ring electrodes (Brain Quick System 98, Micromed, Mogliano Veneto, Italy). The system included a 32-bit A/D converter, a gain of 10 µV/Div, and a band-pass filter ranging from 0.02 Hz to 70 Hz. Electrodes were positioned on the scalp at Fz, Cz, Pz, F3, F4, C3, C4, P3 and P4 locations based on the international 10–20 system. A common reference electrode was placed on the mastoid process in a monopolar montage, with the ground electrode attached at Fpz.

Additionally, four bipolar electrodes were used for electrooculogram recordings: two positioned on the outer canthi to monitor horizontal eye movements and two placed above and below the right eye to detect blinks. Electrode impedances were maintained below 5 kΩ. The signals were sampled at 1024 Hz, and a 50 Hz notch filter was applied to eliminate line noise.

Participants stood comfortably with their arms relaxed at their sides and their eyes open. They were instructed to avoid muscle contractions, head or neck movements, teeth clenching, swallowing or pulling wires to minimise artefacts. Auditory stimuli were delivered through a loudspeaker positioned 1 m behind them, consisting of two sounds with a 2 s interstimulus interval. The first sound (S1) served as a warning stimulus, while the second (S2) acted as a response cue. Both auditory stimuli had a duration of 100 ms, an intensity of 60 dB and a frequency of 2 kHz.

Participants were asked to concentrate on the stimuli and step as quickly as possible using the affected limb in response to S2. To maintain natural behaviour, no specific instructions were given regarding the speed or length of the steps. A total of 50 trials were performed per participant, with a 30 s interval between trials. Data acquisition began 2 s before the warning stimulus.^{7 10 32} Participants completed at least three practice trials prior to the main tests to familiarise themselves with the procedure.

Data analysis was conducted using EEGLAB V.7.1.4, a signal processing platform that separates artefacts from electrocortical signals using independent component analysis.³³ Initially, all epochs were visually inspected, and noisy epochs were excluded. ICA was then applied to eliminate residual artefacts, such as muscle activity, eye blinks and eye movements, while preserving the EEG data.

For analysis, EEG signal epochs were time-locked to the response stimulus, spanning from −2500 to 0 ms, with a 500 ms baseline window before the warning stimulus (−2500 to −2000 ms) (figure 2). Artefact-free signals were baseline corrected and ensemble-averaged for each electrode and subject to improve the signal-to-noise ratio. Next, for each ensemble-averaged signal, the mean amplitude during the 200 ms before the response stimulus was calculated and used as the late CNV amplitude.¹⁹ The maximum amplitude within the warning-response interval was identified as the CNV peak amplitude, and its latency relative to S2 was considered the CNV peak time.¹⁰

Since CNV is most prominent at the Fz, Cz, Pz, C3 and C4 electrodes, data from these sites were analysed statistically. Across all participants, the number of artefact-free epochs per subject ranged from 46 to 50.

Biomechanical outcomes

Participants stood barefoot on a force plate (Bertec, Columbus, Ohio, USA) with their feet angled outward at 10° and their heels separated by 6 cm. Body weight was evenly distributed between both feet. To ensure consistent foot positioning throughout the trials, the outline of each foot was traced onto a sheet of paper and secured to the force plate for the entirety of the test. For data analysis, the raw data were processed with a zero-phase shift, sixth-order Butterworth low-pass filter, with a cut-off frequency of 10 Hz.^{34 35} The duration of the APA phase of GI was defined as the time interval from the onset of centre of pressure (COP) displacement to the most posterior-lateral point of the COP path under the swing limb (figure 3). The onset of COP displacement was identified as the moment when the vertical ground reaction force exceeded the mean plus 2 SDs of the force during the initial 500 ms of quiet stance.^{34 35}

Clinical outcomes

Dynamic balance

The Y-balance test was employed to assess dynamic balance. Participants stood on their injured leg while extending the other leg in three designated directions on the ground: anterior, posterior-medial and posterior-lateral. The posterior-medial and posterior-lateral directions were set at 90° angles to each other, with both forming 135° angles with the anterior direction. Participants were instructed to reach as far as possible with their moving foot in each direction while maintaining balance. The distance from the centre to the farthest point reached was recorded. To normalise the results, the distance was divided by the length of the lower limb (measured from the anterior superior iliac spine to the centre of the medial malleolus) and then multiplied by 100 to express it as a percentage of the lower limb length. Each participant performed the test three times in each direction, and the average distance from the three trials was used for data analysis. Trials were considered invalid if the standing leg was lifted off the ground or if the participant was unable to maintain balance on the support leg during the test. In such cases, the trial was repeated.^{12 13}

Severity of perceived ankle instability

The CAIT was used to evaluate the severity of perceived ankle instability. It contains nine questions, with a maximum score of 30, where higher scores indicate greater ankle stability. The Persian version of this questionnaire has shown good internal consistency (Cronbach’s α of 0.78 for the right ankle and 0.79 for the left ankle) and strong reliability (Intraclass Correlation Coefficient [ICC(2,1)]=0.88; 95% CI 0.86 to 0.90) in athletes.³⁶

Sample size

The sample size was determined using G*Power V.3.1.9.2 software based on a pilot study with 10 subjects, focusing on the late CNV amplitude at Cz. The calculation was based on a within-between interaction effect size of 0.071, derived from preliminary data in the pilot study. This effect size reflected the magnitude of the observed effects, forming the basis for the sample size estimation. The parameters included a power of 0.8, α=0.05, a non-sphericity correction (ε) of 1 and a correlation among repeated measures of 0.5. The analysis indicated that at least 28 participants were required to detect a within-between interaction effect in a design with 2 groups and 2 measurements. To account for a potential 10% dropout rate, 30 participants were recruited.

Randomisation and blinding

Participants were randomly allocated to either the intervention group (balance exercises combined with real tDCS) or the control group (balance exercises combined with sham tDCS) in a 1:1 ratio. Randomisation was conducted through a web-based randomisation service (www.randomization.com) by a third party independent of the study. Block randomisation with a block size of four participants was employed; a technique that organises participants into blocks and then randomly assigns them to different groups within each block. By setting a block size of four, a balanced allocation was maintained by assigning two participants to the intervention group and two to the control group within each block, ensuring consistent group sizes throughout the enrollment process. To ensure allocation concealment from the primary researchers, group assignments were recorded on cards and placed in sequentially numbered, opaque, sealed envelopes. Participants were unaware of their group assignments throughout the study, ensuring a single-blind design. In general, unblinding might have been required if a participant had experienced a serious adverse event following brain stimulation, had developed severe unexpected side effects or had faced a potentially life-threatening condition where knowing the treatment assignment was crucial for immediate medical intervention. In such cases, unblinding would have enabled healthcare providers to make informed decisions regarding the participant’s care. However, in this study, no such circumstances occurred, and all participants remained unaware of their group assignments throughout the trial, maintaining the single-blind design and ensuring unbiased results.

Side effects

The occurrence and severity of various side effects, including itching, tingling, burning, headache, neck pain, skin redness, drowsiness, difficulty concentrating and mood swings, were systematically monitored in all participants. To assess potential discomfort at the electrode sites during or after the intervention, participants completed a questionnaire using a Numeric Analogue Scale, where 0 indicated no sensation and 10 represented the worst imaginable sensation. This scale measured the intensity of reported side effects. Table 2 displays the mean Numeric Sensation Scores for participants in both the real and sham tDCS groups.

Table 2. Mean and SD of Numeric Sensation Scores reported by participants in both groups.

Side effects	Control (N=15)		Intervention (N=15)
Side effects	Mean	SD	Mean	SD
Itching	1.20	0.77	2.07	0.96
Tingling	1.60	0.63	2.20	0.41
Burning	0.80	0.56	0.93	0.80
Headache	0.53	0.52	0.53	0.64
Neck pain	0.00	0.00	0.00	0.00
Skin redness	0.13	0.35	1.27	0.70
Drowsiness	0.07	0.26	0.07	0.26
Difficulty concentrating	0.07	0.26	0.00	0.00
Mood swings	0.00	0.00	0.00	0.00

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Note: 0 represents no sensation, while 10 indicates the worst imaginable sensation.

Statistical analysis

For statistical analysis, IBM SPSS Statistics V.23.0 was used. The Shapiro-Wilk test assessed the normality of the data distribution. Most variables followed a normal distribution, with the exception of time since last sprain and CNV peak time. The Fisher’s exact test compared the proportions of gender and affected side between groups. For normally distributed data, an independent t-test was conducted, while the Mann-Whitney U test was applied to non-normally distributed data.

To analyse the main effects of group (balance exercises+tDCS vs balance exercises+sham tDCS), time (pretreatment and post-treatment), and their interactions, a generalised linear mixed effects model (GLMM) was employed. Group, time and their interaction effects were treated as fixed effects, with participants as random effects. For CNV peak time, a gamma distribution and log link function were used, while a normal distribution and identity link function were applied to other normally distributed variables. When significant interactions were found, the groups were analysed independently to gain more detailed insights. Additionally, partial eta squared (η²) was used to measure effect size, with thresholds indicating small (0.01–0.06), moderate (0.06–0.14) and large (≥0.14) effects.

Additionally, to examine the relationship between treatment-induced changes (post-test minus pretest) in neurophysiological, biomechanical and clinical outcomes, Pearson’s correlation coefficient was used, as the change values followed a normal distribution. These correlations were computed for all participants, regardless of group assignment. The strength of correlation was assessed using the correlation coefficient r, with values between 0.80 and 1.0 indicating a very strong correlation; 0.60–0.79 a strong correlation; 0.40–0.59 a moderate correlation; 0.20–0.39 a weak correlation and values less than 0.20 indicating a negligible correlation.³⁷

Modifications to the registered study protocol

The protocol originally specified a double-blind design, but the final study implemented a single-blind design. Additionally, the protocol listed an age range of 18–35 years, whereas the final study included participants aged 18–40 years. The sample size was initially reported as 46 but was adjusted to 30 based on final calculations. Details and justifications for these modifications are provided in online supplemental file 1.