Evaluating Test-Retest Reliability of Surface Electromyography (sEMG) in Measuring Fatigue in Adolescents Through Prolonged Activity
Phase: NA
Conditions: Healthy Adolescents
Sponsor: Universiteit Antwerpen

Protocol version 1 – 31/10/2024 
 
 
Evaluating Test-Retest Reliability of surface electromyography (sEMG) in Measuring 
Fatigue in Adolescents Through Prolonged Activity 
 
Contents 
1. 
Research Team ................................................................................................................................ 2 
2. 
Background ...................................................................................................................................... 2 
3. 
Aims ................................................................................................................................................. 2 
4. 
Methode .......................................................................................................................................... 3 
1. 
Participants and recruitment: ..................................................................................................... 4 
2. 
Intervention ................................................................................................................................. 5 
3. 
Evaluation .................................................................................................................................... 6 
1.1. 
Anamnese ............................................................................................................................ 6 
1.2. 
Surface electromyography protocol .................................................................................... 6 
4. 
Outcome measures ..................................................................................................................... 7 
5. 
Data management ........................................................................................................................... 7 
6. 
Relevance of the research ............................................................................................................... 7 
References ............................................................................................................................................... 7 
 
 
 
 

Protocol version 1 – 31/10/2024 
 
 
1. Research Team 
Principal Investigator 
Prof. Dr. Ann Hallemans, onderzoeksgroep 
MOVANT & M²OCEAN bewegingslab, Faculteit 
Geneeskunde en Gezondheidswetenschappen, 
Universiteit Antwerpen 
ann.hallemans@uantwerpen.be 
Researchers 
1) Aisyah Naqiyah Binti Rosman 
aisyah.rosman@uantwerpen.be 
 
2) Nathalie Roussels 
nathalie.roussel@uantwerpen.be 
 
3) Elissa Embrechts 
elissa.embrechts@uantwerpen.be 
 
4) Ioanna Nasi 
ioanna.nasi@student.uantwerpen.be 
 
5) Nancy Hassan 
nancy.hassan@student.uantwerpen.be 
 
6) Serena Mane 
serena.mane@student.uantwerpen.be 
 
 
 
 
 
EDGE nummer 
NA 
2. Background 
Muscle fatigue is a multifaceted physiological phenomenon, characterized by a decline in muscle 
performance during sustained or repeated activities (Cifrek et al., 2009). It results from a combination 
of central and peripheral mechanisms, ultimately leading to a reduced ability to maintain force or 
perform tasks. Adolescents engaged in prolonged physical activities, such as sports and dance, may 
experience fatigue, which has the potential to impair performance and could contribute to an 
increased risk of injury (Ratel et al., 2006). Understanding and accurately assessing muscle fatigue is 
therefore essential for optimizing performance, preventing overtraining, and reducing injury risk in this 
population. 
Surface electromyography (sEMG) is a non-invasive technique that measures the electrical activity of 
muscles during contraction (Cao et al., 2017). This method has proven to be effective in detecting 
muscle fatigue by capturing changes in muscle electrical activity that occur as fatigue progresses 
(McManus et al.2020). Typical sEMG changes associated with fatigue include increases in signal 
amplitude, such as the root mean square (RMS) and integrated EMG (iEMG) indicate muscle activation 
intensity (Sun et al., 2022) and decreases in the mean and median frequencies of the sEMG power 
spectrum (Gál-Pottyondy et al., 2023), reflecting alterations in muscle fiber conduction velocity. These 

Protocol version 1 – 31/10/2024 
 
 
sEMG characteristics have been widely studied in adults, but their application and reliability in 
adolescents, especially during prolonged activities, remain underexplored. 
Adolescents, due to their unique physiological development, present specific challenges for muscle 
fatigue assessment (Patikas et al., 2018). Given their ongoing growth, differences in muscle fiber 
composition, and varying levels of physical maturity, muscle fatigue patterns in adolescents may differ 
from those in adults (Bontemps et al., 2019). It is crucial to establish reliable tools for assessing muscle 
fatigue in this age group, as subjective fatigue reporting may be inconsistent or inaccurate. Reliable 
objective measures like sEMG could provide critical insights into fatigue management and 
performance optimization for young athletes. 
To be a valuable tool in fatigue assessment, sEMG must demonstrate both test-retest and inter-rater 
reliability. Test-retest reliability refers to the consistency of measurements over time when the same 
participants are tested under similar conditions on different occasions (Lynn et al., 2018), while inter-
rater reliability examines the consistency of measurements when different raters conduct the 
assessments (Mchugh, 2012). Establishing both forms of reliability is essential to ensure that sEMG can 
be reliably used to assess muscle fatigue in adolescents without being influenced by measurement 
error or variability introduced by different assessors. 
Aims 
To determine the test-retest reliability of surface electromyography (sEMG) outcomes in detecting 
levels of fatigue among healthy adolescents. This study will examine the consistency of sEMG readings 
across repetition of the exercises and assess how these measurements correlate with performance 
metrics and subjective fatigue assessments. 
We formulate the following hypotheses: 
1. sEMG will demonstrate high test-retest reliability in measuring fatigue levels across two sessions 
of prolonged activity in healthy adolescents, demonstrating its consistency as a tool for fatigue 
assessment. 
2. The patterns of fatigue detected by sEMG will correlate with subjective measures of fatigue and 
performance decline, supporting the tool's construct validity in capturing the multifaceted nature 
of fatigue in this population.  
3. Method 
Cross-sectional study with test- retest design 
Duration: one week per participant. 

Protocol version 1 – 31/10/2024 
 
 
 
 
Figuur 1: Schematische weergave van het verloop van de studieA 
 
1. Participants and recruitment: 
- Population: Healthy adolescents aged 13.0-18.9 years will be recruited for the study. To be included, 
participants can have no known neuromuscular disorders or conditions that could affect muscle 
function. Participants can be physically active for up to 60 minutes per day, on average (Piercy et al., 
2018) but cannot be involved in any specific, rigorous training programs towards elite competition 
sports. Adolescents will be recruited from local schools, sports clubs, and among the children of 
University of Antwerp staff members. 
Adolescents, Age: 13 y 0  months-
18 y 11 months
Informed consent of participants 
and legal representative
Session 1
Pre-fatigue Evaluation: sEmg 
baseline reading (quadriceps and 
gluteus muscles);  Borg- scale (10 
point) to access perceived 
exertion before fatigue; ROM 
assessment for squats 
Intervention: Repetitive 
bodyweight squats until could not 
maintain full ROM
Post-Fatigue evaluation: sEMG 
reading post-exercise; Borg-sclae 
(10 point) to access perceived 
exertion after fatigue; assessment 
of performance decline based on 
ROM during squats
Session 2 
(after 1 week)
Pre-fatigue
Intervention
Post-fatigue
Exclusion: vigorous physical 
activity < 48h, medical condition 
affecting muscle function; Heavy 
training/ competiiton = 1 week 
waiting time

Protocol version 1 – 31/10/2024 
 
 
- Sample Size: 27 participants to account for potential dropouts and ensure robust statistical power. 
For the sample size of this study, we selected the intraclass correlation coefficient (ICC) value of 0.7 as 
the target for reliability analysis, based on the “consensus-based cut-off point for sufficient reliability” 
(Mokkink et al., 2023). The sample size determination was performed using an alpha level of 0.05 and 
a medium effect size of 0.25 (Huang et al., 2023), which reflects a moderate relationship between test 
sessions. 
The power analysis indicated that 21 participants would provide a statistical power of 0.80 to detect 
significant test-retest reliability, according to the COSMIN Checklist (Mokkink et al., 2023). However, 
to account for a 20% potential dropout rate, the final target sample size has been increased to 27 
participants. This calculation assumes two repeated measurements per participant and a single group 
design. As ICC estimation generally requires a smaller sample size, this is appropriate for our pilot 
study, where test-retest reliability is being assessed in an initial, limited participant pool (Tan et al., 
2013). 
- Inclusion Criteria: Healthy adolescents with no known neuromuscular disorders. They should be 
engaged in regular physical activity, defined as participating in normal physical activity for up to 60 
minutes per day, and must not be involved in any specific or rigorous training programs for elite sports. 
- Exclusion Criteria: Adolescents with any medical condition affecting muscle function, or those 
currently on medications influencing muscle performance. Additionally, participants who have 
engaged in intense physical activities within the 48 hours prior to the study will be excluded. If 
participants have taken part in heavy training or competition, they will be required to wait a minimum 
of one week before being eligible to complete the second session of the study. 
2. Intervention 
To induce muscular fatigue, participants will perform a repetitive bodyweight squat until 1) the squat 
cannot be performed over the full range of motion for 2 consecutive repetitions and 2) physical 
exertion occurs (10-point Borg scale). Squats were chosen as they effectively engage both muscle 
groups (quadriceps and gluteus muscles) and have been proven to induce fatigue over time. The 
exercise will be performed at a predefined tempo using a metronome to maintain a consistent 
repetition rate, ensuring controlled and uniform movement across participants. The squats will 
continue until participants are unable to achieve the full range of motion (ROM) for two consecutive 
repetitions or are unable to maintain the tempo set by the metronome.  
During the activity, sEMG measurements will be taken to assess muscle activation and fatigue levels in 
the quadriceps and gluteus muscles. The combination of objective sEMG data and subjective fatigue 
ratings will allow for a comprehensive assessment of fatigue. 
Each participant will visit the laboratory twice over the course of the study. During the first visit, 
participants will complete a pre-fatigue evaluation, which includes baseline sEMG recordings, ROM-
assessment, and perceived exertion measured via the Borg scale. The intervention (squats) will be 
performed following these initial assessments. Post-intervention, another evaluation will be 
conducted, including sEMG recordings, ROM reassessment, and post-exercise Borg scale ratings to 
assess performance decline and perceived fatigue. 
Participants will undergo a second session one week later to evaluate the test-retest reliability of sEMG 
in assessing muscle activation patterns and fatigue levels. The second session will replicate the first, 
with sEMG measurements taken continuously to assess the consistency of muscle activation patterns, 
performance decline, and perceived fatigue across the two sessions. This data will be used to verify 

Protocol version 1 – 31/10/2024 
 
 
the high test-retest reliability of sEMG and to evaluate intra-rater reliability, ensuring minimal 
variability in measurements between different assessors and across sessions. 
Throughout both sessions, sEMG readings will be recorded continuously to evaluate the consistency 
of muscle activation patterns and their relationship to performance decline and perceived fatigue 
levels. The collected data will be analysed to verify the high test-retest reliability and intra-rater 
reliability of sEMG as hypothesized, with minimal variability across sessions. 
 
3. Evaluation 
Duurtijd: 90 – 120 minuten 
1.1. Anamnese  
During the initial assessment, participants will be asked a series of questions to gather information 
about their general health status and relevant background. The following aspects will be covered: 
- Age: Participants will be asked to provide their age and date of birth, to ensure they fall within the 
inclusion criteria of 13.0-18.9 years. 
- Height and Weight: Basic anthropometric measurements, including height, weight and leg length will 
be recorded to assess the participants' physical characteristics. 
- Sports Participation: Participants will be asked using physical activity questionnaires for adolescents 
(PAQ-A) questionnaires about the types of physical activities they are engaged in, including the 
frequency and duration of participation in sports, dance, or other physical activities. This will help 
confirm that they meet the requirement of engaging in normal physical activity and are not involved 
in any structured or rigorous training programs. 
- Health Status: A series of health status questionnaires will be asked to determine whether 
participants have any known neuromuscular disorders, medical conditions, or injuries that could affect 
muscle function. Participants will also be asked if they are currently taking any medications that might 
influence muscle performance. 
1.2. Surface electromyography protocol 
- Baseline Assessment: Setup and calibration of sEMG sensors will be conducted on the identified 
muscle groups, placed bilaterally on both the left and right sides of the body. The primary lower limb 
muscles to be monitored include the gluteus medius, rectus femoris, vastus lateralis (quadriceps), 
biceps femoris (hamstrings), tibialis anterior, and gastrocnemius medialis (calves). This setup fully 
utilizes the 16-channel sEMG system by monitoring each muscle group bilaterally to capture 
comprehensive data on muscle activity and fatigue. Before the intervention, participants will undergo 
a baseline evaluation that includes sEMG recordings of muscle activation and an assessment of ROM 
during squats. 
- Activity Measurement: sEMG measurements  and perceived exertion using the Borg Rating of 
Perceived Exertion scale will be taken during the fatigue-inducing exercises (full-bodyweight squats). 
Immediately after the squats, participants will walk for a short duration to assess post-fatigue muscle 
activity. sEMG measurements will be taken during this walking phase to capture immediate post-
fatigue muscle activation in the quadriceps and gluteus muscles.  
- Follow-Up Measurement: The entire protocol will be repeated after a recovery period of a week, 
under identical conditions. This will allow for the assessment of the test-retest reliability of the sEMG 

Protocol version 1 – 31/10/2024 
 
 
data, ensuring that muscle activation patterns, as well as the overall protocol, remain consistent and 
reliable across different testing sessions.  
4. Outcome measures 
Descriptive  parameters 
Demographics: age (years), gender, PAQ-A score 
Anthropometric data: height (cm), weight (kg), BMI, leg length (cm) 
Primary Outcome measure: sEMG median power frequency (MPF) and root mean square (RMS) 
Secondary Outcome measures: Borg RPE score 
The statistical analyses for this study will be conducted using IBM SPSS Statistics 29 with G*Power 3.1, 
allowing for comprehensive data analysis, including descriptive statistics, reliability testing, and 
inferential statistics to explore the relationships between variables of interest and to compare 
outcomes across sessions and groups. 
- Descriptive Statistics: These will summarize participant characteristics and sEMG outcome measures, 
including means, standard deviations (SD), and ranges. We'll calculate means and standard deviations 
for sEMG median power frequency (MPF) and root mean square (RMS) at each trial and visualize sEMG 
patterns across the gait cycle using ensemble averages and standard deviation. 
- Inferential Statistics: The ICC analysis will provide a statistical measure of how consistent the sEMG 
readings are between sessions (e.g., test and retest) for each. A two-way mixed effect, absolute 
agreement, multiple raters/measurements ICC model will be used, with ICC values ≥ 0.75 indicating 
good to excellent reliability and values between 0.6-0.74 indicating good reliability. Confidence 
intervals for ICC estimates will also be calculated. Additionally, Bland-Altman plots will be used to 
identify any systematic bias and outliers in test-retest measurements.  
4. Data management 
The data obtained from the various tests are coded (pseudonymised) and processed with Matlab, 
Excel, SPSS and JMP to design tables (.xlsx) and figures (.pdf, .eps, .bmp, .pptx). Encrypted data is also 
entered into a REDCap database via an eCRF. Data that is stored for a long time (in the secure 
institutional repositories) contains only encrypted data. Data protection procedures according to the 
GDPR (including GCP) will be applied to comply with national and EU legislation on the protection of 
the processing of personal data. 
5. Relevance of the research 
This research aims to enhance the understanding of how muscle fatigue can be reliably measured using 
sEMG in adolescents. Findings could lead to improved protocols for monitoring and managing fatigue 
in young athletes, potentially influencing training routines and performance enhancement strategies. 
 

Protocol version 1 – 31/10/2024 
 
 
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