Study design

This study protocol describes a two-arm, parallel-group randomized controlled trial (RCT) that will employ equal allocation (1:1) to evaluate the effects of an eight-week neuromuscular-cognitive training protocol on ACL injury risk factors in female volleyball athletes with DKV. Ethical approval was granted by the Research Ethics Committee of Bu-Ali Sina University (IR.BASU.REC.1402.076). This study has been registered with the Iranian Registry of Clinical Trials (IRCT20190224042827N5), registration date: 24 March 2024. The protocol integrates neuromuscular training principles [18], a cognitive intervention design model for ACL prevention [19], and the JUMP-ACL framework [20] to concurrently enhance motor and cognitive functions, with the aim of improving athletes’ adaptability in competitive environments where physical skills and rapid cognitive processing are critical. Assessments will be conducted at baseline (pre-intervention) and immediately following the eight-week intervention (post-intervention) to evaluate the protocol’s impact on primary and secondary outcomes.

Setting

The study will be conducted among professional and collegiate female volleyball teams in Hamedan Province, Iran. All screening, training sessions, and outcome assessments will be performed in the Sports Rehabilitation Laboratory at Bu-Ali Sina University, equipped with 2D motion analysis systems, a foot scan plate, EEG and EMG equipment, and supervised by experienced sports medicine and rehabilitation professionals.

Participants

Maintenance strategy

Several strategies will be used to minimize participant dropout. A study coordinator will send reminder text messages before each scheduled training and assessment session. Training sessions will be embedded within each team’s existing practice schedule to minimize scheduling conflicts with competitions and academic commitments. Where feasible, makeup sessions will be offered for absences due to illness or minor scheduling conflicts, provided the participant does not meet the withdrawal threshold defined in the Exclusion Criteria. Reasons for dropout or withdrawal will be systematically recorded and reported using a CONSORT flow diagram. Participants who complete the full protocol will receive individualized feedback on their landing mechanics as a non-monetary incentive to continue engaging.

Screening procedures

All potential participants will complete two screening assessments in the Sports Rehabilitation Laboratory:

1- Tuck Jump Test with Cognitive Task: Participants will perform continuous tuck jumps for 10 seconds while responding to visual stimuli. The test will be video-recorded and assessed for neuromuscular deficits associated with DKV [21].

2- Single-Leg Landing Task with Cognitive Task: Participants will perform single-leg landings from a 30-cm box while completing a visual discrimination task. Landing mechanics will be captured using 2D motion analysis techniques to quantify knee valgus angles. DKV will be defined as a peak knee valgus angle greater than 12 degrees during the single-leg landing task, consistent with established thresholds for increased ACL injury risk [22].

Inclusion criteria

Participants must meet all of the following criteria:

• Female volleyball players aged 18–23 years
• Minimum of three years of competitive volleyball experience
• Currently training or competing at the club or collegiate level (minimum three training sessions per week)
• Presence of DKV confirmed by screening tests
• No history of ACL injury or reconstruction in either knee
• No lower extremity injury in the past six months requiring medical attention or time away from sport
• Able and willing to participate in training sessions three times per week for eight consecutive weeks
• Able to attend all assessment sessions

Exclusion criteria

Participants will be excluded if they meet any of the following:

• History of ACL reconstruction or other knee ligament surgery
• Current lower extremity injury or pain that limits participation in volleyball activities
• History of concussion within the past six months
• Current participation in other structured injury prevention or cognitive training programs
• Unable or unwilling to provide informed consent
• Missing two consecutive or three non-consecutive scheduled training sessions.

Informed consent

All eligible participants will receive detailed verbal and written information about the study objectives, procedures, potential risks and benefits, and their right to withdraw at any time without penalty. Written informed consent will be obtained from all participants before enrollment. All procedures involving participants in this study will follow the guidelines of the Research Ethics Committee of Bu-Ali Sina University (IR.BASU.REC.1402.076) and adhere to the 2013 Declaration of Helsinki.

Qualifications of intervention providers

The intervention will be overseen by a PhD-level sports rehabilitation specialist with expertise in sports training, who will explain the study’s objectives to coaches and athletes, obtain informed consent, and supervise all assessments in the Sports Rehabilitation Laboratory at Bu-Ali Sina University. Day-to-day implementation of the training program will be conducted by trained team coaches under the supervision of the research team.

Theoretical framework and design principles

This intervention protocol was developed based on the seven-step JUMP-ACL framework, incorporating evidence-based neuromuscular and cognitive training principles [18–20]. The intervention aims to address modifiable neuromuscular and biomechanical risk factors associated with ACL injury in young female volleyball athletes through a structured, progressive eight-week program. Previous literature has suggested that female volleyball athletes demonstrating DKV patterns may exhibit altered lower-extremity biomechanics associated with increased ACL loading during high-demand athletic tasks. Accordingly, the intervention is designed to improve movement mechanics and enhance lower-limb neuromuscular control under cognitively demanding conditions that approximate the attentional demands of real-game scenarios.

Based on previous literature regarding barriers to injury prevention program implementation in team sports and practical considerations identified through consultation with volleyball coaches, potential barriers to adherence include limited awareness of injury prevention strategies, time constraints within training schedules, and limited coach training in neuromuscular-cognitive intervention delivery. To address these barriers, the current protocol incorporates several implementation strategies, including comprehensive coach education, integration of training within existing team schedules, provision of detailed instructional materials, and emphasis on the cognitive training component to enhance athlete motivation and engagement.

Before intervention commencement, a two-hour standardized training workshop will be conducted for all participating team coaches and assistant coaches. The workshop will cover ACL injury mechanisms and prevention strategies, neuromuscular and cognitive contributors to injury risk in female athletes, demonstration and hands-on practice of intervention exercises, administration of cognitive tasks and integration of dual-task conditions, use of standardized performance evaluation checklists, identification of movement and cognitive errors, safety procedures, injury management and communication protocols, and strategies to maintain athlete motivation and adherence.

Following the workshop, coaches will be required to achieve a predefined competency threshold of 80% on a practical skills assessment, demonstrating adequate proficiency in exercise instruction, error identification, and progression decision-making.

Intervention components

The training program combines two main components: neuromuscular training and cognitive training, both designed to address modifiable neuromuscular and biomechanical factors associated with ACL injury risk. Neuromuscular exercises aim to improve dynamic balance, increase strength of key lower limb muscles (especially hip abductors, quadriceps, and hamstrings), enhance proprioceptive function, and refine neuromuscular control during volleyball-specific high-risk movements such as jumping, landing, and cutting. Cognitive exercises are incorporated throughout neuromuscular training to create dual-task scenarios that challenge attention and decision-making under simultaneous cognitive and motor demands.

The cognitive training focuses on four critical areas essential for athletic performance. First, response selection and decision-making speed help athletes respond quickly to visual and auditory cues while performing movements, requiring rapid information processing and appropriate response selection. Second, working memory is trained by requiring athletes to recall movement sequences, task rules, or stimulus-response associations during exercises. Third, response inhibition involves practicing movement choices and inhibitions based on specific cues, requiring athletes to respond to target stimuli while suppressing irrelevant responses. Lastly, athletes train divided attention by monitoring environmental cues, such as volleyball or teammate movements, while maintaining postural control and executing dynamic exercises.

These cognitive exercises are designed to be simple, practical, progressive, and relevant to volleyball-specific demands. They are performed in pairs to facilitate peer feedback and encourage rapid and accurate responses.

All exercises in the program follow four core principles to ensure effectiveness and safety. The first is progressive overload, involving gradual increases in difficulty from simple to complex movements, single-task to dual-task conditions, and stable to less stable conditions when appropriate. The second is variety, as a wide range of exercises is included to maintain athlete engagement and target multiple performance aspects, such as strength, balance, power, and coordination. The third is repetition and continuity, ensuring sufficient practice volume and consistency to reinforce motor learning and potentially facilitate neural adaptations related to motor control. Lastly, expert supervision is essential, with trained staff consistently monitoring exercise execution to ensure proper technique, reduce inappropriate movement strategies, and provide immediate feedback and correction.

Intervention protocol

The intervention will be conducted over eight consecutive weeks, with three supervised training sessions per week, each lasting 60 minutes (a total of 24 supervised sessions). These will be scheduled within the teams’ regular training times to minimize disruption and enhance adherence. Sessions will be performed on non-consecutive days to allow adequate recovery. Each session will follow a standardized structure: Warm-up (15 minutes), including dynamic stretching, light aerobic activity, and sport-specific movements according to established protocols; main training (30–40 minutes), focusing on progressive neuromuscular and cognitive exercises; and cool-down (10 minutes), consisting of static stretching of the primary lower-extremity muscles and breathing exercises to facilitate recovery.

The intervention was structured as a four-phase progressive training protocol, consisting of consecutive two-week stages with specific objectives and gradual increases in motor complexity and cognitive demands (Tables 1 and 2).

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Table 1. Categorization of exercises based on movement plane, limb involvement, and equipment use.

https://doi.org/10.1371/journal.pone.0353944.t001

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Table 2. Description of physical exercises with cognitive tasks of study.

https://doi.org/10.1371/journal.pone.0353944.t002

In the first phase (Weeks 1–2), referred to as the Controlled Movement Phase, the primary goal was to establish proper multi-plane movement patterns and improve movement strategies associated with dynamic knee valgus through foundational exercises performed under high levels of external feedback. Training emphasized sagittal-plane movements with controlled frontal-plane components, simple single-task neuromuscular exercises accompanied by real-time verbal and visual feedback, and the introduction of basic cognitive tasks. Exercises were performed in pairs to allow peer observation and feedback, mainly on stable surfaces with bilateral support when needed, and at low to moderate intensity.

The second phase (Weeks 3–4), referred to as the Strategic Planning Phase, aimed to introduce moderate cognitive demands by emphasizing decision-making and motor planning during movement. This stage incorporated a greater proportion of single-leg exercises, integration of dual-task conditions combining motor and cognitive demands, and more complex cognitive challenges such as choice reaction time and stimulus discrimination. External feedback was reduced to encourage reliance on internal sensory feedback mechanisms, while unstable surfaces and dynamic balance challenges were introduced. Exercises were performed at moderate intensity and complexity.

In the third phase (Weeks 5–6), called the Automatic Processing Phase, the objective was to develop more efficient motor control under cognitive load and improve dual-task proficiency. Athletes received minimal external feedback and were encouraged to self-monitor and correct their performance. Training involved complex multi-directional movements across multiple planes, high cognitive load tasks requiring divided attention, and increased speed and intensity. Sport-specific movement patterns resembling volleyball actions were emphasized, with a focus on efficient and fluid movement execution.

Finally, the fourth phase (Weeks 7–8), designated as the Transfer and Stabilization Phase, sought to facilitate the transfer of acquired skills to competition-like situations, emphasizing frontal-plane single-leg control and reinforcement of learned motor adaptations. This stage involved maximum cognitive load with unpredictable, game-like stimulus conditions, frontal-plane dominant single-leg tasks mimicking volleyball-specific movements such as lateral cutting and defensive slides, and integration of volleyball equipment and scenarios. Exercises were performed at high intensity and speed, under fatigue and time pressure, to facilitate application of learned skills during dynamic sport situations.

Athletes will progress to the next training phase only when they consistently meet predefined progression criteria across three consecutive sessions. Movement quality will be assessed by a trained coach using a standardized checklist, ensuring appropriate lower-extremity alignment according to predefined movement-quality criteria. Cognitive performance will also be evaluated, requiring athletes to commit no more than three predefined cognitive errors per session during tasks involving stimulus responses or rule-based challenges. Athletes must maintain movement quality under dual-task conditions without clinically meaningful deterioration compared with single-task performance and execute tasks at the prescribed speed and intensity while meeting cognitive demands. Since progression is individualized, athletes who do not meet the criteria after completing the planned two-week phase will remain at the same level with modified or alternative exercises of similar complexity until the requirements are achieved, thereby supporting adequate motor learning, confidence development, and minimizing inappropriate progression-related injury risk. All progression decisions will be documented using standardized forms and reviewed weekly by the supervising sports rehabilitation specialist to ensure safety.

Training intensity will be indirectly quantified using the session rating of perceived exertion (session-RPE) method [23]. Approximately 30 minutes after each session, each participant will rate her perceived exertion using the modified Borg CR-10 scale; session-RPE will then be multiplied by session duration (minutes) to calculate session training load. Weekly training load will be reviewed by the supervising sports rehabilitation specialist alongside the movement-quality and cognitive-error checklists to identify unusual increases in perceived exertion and inform individualized progression decisions.

Control group protocol

The control group will receive an active control intervention consisting of the same neuromuscular training exercises as the experimental group but performed exclusively under single-task conditions without cognitive challenges. This design will control for exercise volume, intensity, trainer attention, and social interaction, thereby estimating the additional contribution of cognitive training integration. Training will be delivered three times per week for eight weeks, with each session lasting approximately 60 minutes and following an identical structure to the experimental group: a 15-minute warm-up, 30–40 minutes of main training, and a 10-minute cool-down. Progression will follow the same four-phase structure, with gradual increases in exercise difficulty, intensity, and complexity. Instructional methods will include verbal cueing, demonstration, and corrective feedback; all will be provided with a single-task focus to ensure consistency across groups while eliminating additional cognitive demands.

Both groups will continue their regular volleyball team training sessions, competitions, and usual physical activities throughout the 8-week intervention period. All participants will be instructed to maintain consistent training and competition schedules and report any changes in activity level, injuries, or other relevant health factors.

To enhance motivation and adherence to the eight-week neuromuscular-cognitive training program, each female volleyball player will set an individualized goal based on her personal objectives. These goals will be used to enhance engagement without altering the standardized exercise prescription. A simple checklist will be provided to each participant to track progress, monitor ongoing performance, and record the frequency and type of cognitive and neuromuscular errors, encouraging motivation through ongoing personalized feedback. To avoid mental fatigue and maintain engagement, the protocol includes varied neuromuscular and cognitive training components. Team coaches will actively participate in delivering the training, guiding sessions, and ensuring high-quality execution.

During the intervention period, participants will be instructed to avoid performance-enhancing stimulants, tobacco, non-steroidal anti-inflammatory drugs (NSAIDs), or other analgesics within 24 hours before each training session to minimize potential effects on pain perception and physical performance. They will be permitted to continue their regular team training sessions and maintain their usual physical activity and dietary habits throughout the study.

Timeline

The study timeline and schematic overview, including participant enrolment procedures, baseline and post-intervention outcome assessments, and the eight-week intervention period, are presented in Fig 1.

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Fig 1. Schedule of enrolment, interventions, and assessments.

 − t₁, enrolment and screening week; t₀, allocation and baseline assessment; t₁ - t₃, interventions; t₄, post-interventions assessment.

https://doi.org/10.1371/journal.pone.0353944.g001

Outcomes

Participants will first receive detailed information about the study procedures, ethical considerations, and potential risks before providing informed consent and completing a personal information form. They will then undergo standardized instructions and a familiarization session, followed by a warm-up to standardize physical readiness and minimize injury risk. Assessments will be conducted 72 hours before the start of the eight-week neuromuscular-cognitive training protocol (pre-test) and within 72 hours after the final session (post-test) to evaluate its effects on modifiable ACL injury-related risk factors in female volleyball players with DKV. All assessments will take place in the Sports Rehabilitation Laboratory at Bu-Ali Sina University under standardized temporal and environmental conditions, with precise measures to control external variables and ensure accurate evaluation.

The primary outcome is the change in two-dimensional landing kinematics, particularly movement patterns associated with DKV, during single-leg landing under two conditions: with and without a concurrent cognitive task. Secondary outcomes include electrocortical brain activity measured by electroencephalography (EEG), lower limb muscle activity assessed by surface electromyography (sEMG), single-leg balance, lower limb muscle strength, and knee joint proprioception. These measures will determine the intervention’s impact on neuromuscular, cognitive, and biomechanical factors associated with ACL injury risk.

Harms

All self-reported adverse events, unintended intervention effects, or changes in participants’ health status, including pain, discomfort, musculoskeletal symptoms, or injuries, will be documented and reported to the principal investigator for review and appropriate action.

Sample size

The sample size was calculated using G*Power software (version 3.1) for a repeated-measures ANOVA (within-between interaction) to detect changes in DKV angle at initial contact. The effect size was derived from the group × time interaction reported for DKV at initial contact in a previous neuromuscular training study with dual cognitive tasks [24] (partial η² = 0.19), converted to an effect size f = 0.48. Based on a significance level (α) of 0.05, statistical power (1-β) of 0.95, and an assumed correlation of r = 0.5 between repeated measures, a minimum total of 18 participants was required. The required sample size was initially determined based on the primary outcome measure (the DKV angle) to ensure sufficient statistical power. To adjust for multiple secondary outcome assessments and an estimated 20% attrition rate, we inflated the target enrollment to 30 participants, who will be randomly allocated in a 1:1 ratio to the intervention and control groups.

Assignment of interventions: Allocation

Concealment mechanism

To conceal allocation and prevent selection bias, the randomization sequence will be sealed in opaque, tamper-proof, sequentially numbered envelopes. Each envelope will contain a card indicating group assignment corresponding to the participant’s number. The envelopes will be opened only after completion of baseline assessments. An independent individual, not involved in study implementation, will prepare and manage the envelopes.

Implementation

An independent investigator, not involved in participant recruitment, assessment, or intervention administration, will generate the randomization sequence and maintain exclusive access to the randomization list. After confirming eligibility and obtaining informed consent, this investigator will randomize participants. The study coordinator, blinded to group assignment, will enroll eligible female volleyball athletes with DKV and schedule assessments. Due to the nature of the intervention, trainers cannot be blinded to group allocation. The allocation process is designed to minimize selection and performance bias throughout the study.

Assignment of interventions: Blinding

This study will be conducted as a participant- and assessor-blinded randomized controlled trial. Participants will be informed that they are receiving training, but will not know whether the program includes neuromuscular training with or without cognitive components. Given the structural similarity between protocols, participants may be unlikely to determine their group assignment. To minimize communication between groups, intervention and control sessions will be scheduled on separate training days. Outcome assessors will remain blinded to group assignments. Participants will be instructed not to disclose intervention-related information during evaluations.

Data collection methods

To evaluate landing kinematics, electrocortical brain activity, and muscle activity, a single-leg landing test will be administered under two experimental conditions: with and without a concurrent cognitive task. After reflective markers and EMG sensors are placed on the participant’s preferred kicking leg (determined using a ball-kicking test) and the EEG cap is fitted, participants will perform the landing test three times with cognitive load and three times without cognitive load. During the test, frontal-plane knee valgus and sagittal-plane knee flexion angles, as well as trunk flexion and lateral flexion angles, will be captured using two cameras positioned for anterior and lateral views. Muscle activity in the vastus medialis, vastus lateralis, biceps femoris, semitendinosus, and gluteus medius will be measured using surface EMG. Electrocortical brain activity will be recorded via 19 EEG channels at theta and alpha-2 frequency bands [25].

To begin the test, participants will cross their arms over their chest to minimize upper-limb movement while standing on a 30 cm-high step [22]. The heel of the dominant foot will be positioned at the edge of the step to limit preparatory movement. Participants will look forward to a wall-mounted screen. After the presentation of the final arrow cue, the participant will perform a single-leg landing on either the right or left target area marked on the floor, based on the direction of the final arrow. The test will also be conducted without cognitive load as a control condition. If the participant cannot maintain balance or demonstrates loss of control (for example, requires additional support, steps outside the target area, or loses postural stability), the trial will be considered invalid and excluded from analysis [26].

After completing the landing tests, participants will stand on a foot scan platform to perform a 30-second balance assessment under eyes-open and eyes-closed conditions. Subsequently, knee muscle strength (flexors and extensors) and hip muscle strength (abductors and adductors) will be measured using a hand-held dynamometer [17]. Knee joint proprioception at 60° of flexion (non-weight-bearing) will be assessed using a goniometer.

In addition, health-related quality of life will be evaluated before and after the intervention using the SF-12 questionnaire [27]. The analysis criterion for all variables will be the change from baseline values.

Data management

Two trained sports rehabilitation specialists will collect and enter all data electronically using secure, password-protected software. Each participant will receive a unique identification number to ensure anonymity. The principal investigator (PI), in collaboration with the data management and analysis team, will perform data quality control, checking for missing values, out-of-range entries, and duplicate records. This team will operate separately from the intervention delivery team to minimize potential bias and ensure data integrity, adherence to pre-specified statistical procedures, and reproducibility.

Statistical methods

Descriptive statistics, including means and standard deviations, will summarize the data. The Shapiro-Wilk test will assess data normality. Baseline characteristics will be summarized descriptively between groups. Repeated-measures ANOVA will be used to evaluate the group × time interaction effect as the primary indicator of intervention effectiveness (training vs. control; pre-test vs. post-test). Pairwise comparisons following significant interactions will be conducted with Bonferroni correction to control type I error. The five secondary outcome domains (cortical brain activity, lower-limb muscle activation, single-leg balance, muscle strength, and knee joint proprioception) will be interpreted as exploratory outcomes and hypothesis-generating findings. Partial eta squared will quantify effect sizes, interpreted as small (0.01), medium (0.06), or large (0.14). All analyses will be performed using SPSS version 27 with α = 0.05 and a 95% confidence level.

Given the modest sample size, supplementary analyses will be conducted. Baseline values will be reviewed descriptively, and if clinically meaningful baseline imbalance is observed, ANCOVA models adjusting for baseline values will be performed as sensitivity analyses. Missing data patterns will be assessed, and linear mixed models with appropriate covariance structures will be considered as sensitivity analyses to evaluate robustness under missing-at-random assumptions. Any meaningful discrepancy between supplementary analyses and the primary ANOVA will be reported and discussed.

To assess clinical significance and intervention effectiveness, minimal clinically important difference (MCID) and minimal detectable change (MDC) will be calculated at a 95% confidence level. The MDC will be derived using the standard error of measurement (SEM), as defined by the following formulas [28]:

The reliable change index (RCI) will be calculated to determine whether observed changes exceed measurement error. In these equations, SDpre denotes the standard deviation of pre-test scores in the training group, and ICC denotes the intraclass correlation coefficient (test-retest reliability). MCID_RCI will be used to determine the minimum clinically significant change. Reliability coefficients from previously reported studies in comparable populations will be used.

Dissemination plans

Results will be disseminated through peer-reviewed journals, including open-access platforms when possible, to ensure broad accessibility for researchers, clinicians, and relevant stakeholders. A plain-language summary, accompanied by visual materials outlining key findings and practical implications, will be shared with study participants and their coaches to support the translation of research findings into sports practice.