Study Design
A short, double-blind, randomized, placebo-controlled clinical trial was prospectively registered on ClinicalTrials.gov (NCT05344183; registered on 25/04/2022) under the name ‘Immediate and Short-term Effects of Low-level Laser'. This protocol was developed based on the SPIRIT statement.
Participants and Recruitment
The study will take place at the Physiotherapy Department of the Federal University of Amapá. Participants will be recruited through a registration form on the Google Forms platform. This study was approved by the Ethics and Research Committee of the Federal University of Amapá (registration number: 58174622.2.0000.0003) and followed all the recommendations of the Declaration of Helsinki. The participants will receive oral and written instructions on the procedures and objectives, and must sign an informed consent form, stating that they agree to participate in the study. All methods were carried out in accordance with relevant guidelines and regulations. At any moment, participants who wish to discontinue their involvement in the study may do so.
An individual evaluation form will be created for each participant, including personal information, anthropometric data, evaluation of functional performance, and electromyographic activity of the quadriceps muscles. All evaluations will be conducted by a blinded evaluator (researcher 01).
Baseline assessment
Participants will complete an evaluation form containing personal and anthropometric data, including gender, age, weight, height, dominant limb, sports modality, frequency of physical and sports activity, and presence or absence of pain complaints, with no history of injury in the last two months [18]. The time point of each assessment can be observed in Table 01.
The study incorporates specific inclusion criteria to ensure a targeted and representative sample of
athletes. Participants must meet the following requirements:
1. Sport and Age: Handball and volleyball athletes aged between 18 and 35 years.
2. Gender: Both male and female athletes are eligible for inclusion.
3. Training Routine: Athletes must maintain a consistent training routine for their respective sport, engaging in training sessions at least twice a week.
Exclusion Criteria: The following conditions will lead to exclusion from the study:
1. Recent Lower Limb Injury: Athletes with a history of acute lower limb injury within the last 2 months preceding the study.
2. Ongoing Pain or Injury: Athletes currently experiencing pain or injury during the study period.
3. Changes in Physical Activity Routine: Participants who undergo any alterations in their regular physical activity routine during the week of evaluations.
By implementing these carefully defined inclusion and exclusion criteria, the study aims to ensure the relevance and reliability of the gathered data and the overall integrity of the research outcomes.
Fatigue Protocol
After the baseline assessment, participants will undergo exercise-induced muscle fatigue using a plyometric protocol modified and adapted for this study based on the protocol suggested by Marginson et al. (2005) [19]. The muscle fatigue protocol will consist of 5 sessions over 5 days (see Fig. 1), each of which will involve 3 sets of 30 repetitions of jump squats with a 1-minute rest period between each set. Before beginning the fatigue protocol, participants will receive standardized instructions and perform familiarization exercises. During the protocol, a therapist will provide verbal guidance and encouragement, and participants will be asked to repeat the exercise if done incorrectly [20]. All participants will perform the same protocol and be guided by the same therapist.
Figure 1. Flowchart of the study. Showing the flow of participants from each group.
Functional Performance
For the evaluation of functional performance (primary outcome) will be evaluated using the unipodal horizontal jump test, a sub-item of the Hop test. Participants will perform a unipodal horizontal jump within an area previously demarcated in centimeters, with three jumps performed on the dominant lower limb. The average distance (in centimeters) of the three jumps will be used for statistical analysis [21].
Electromyographic activity
Muscle activations will be collected using a FREEEMG 1000 (Kinetc, Brazil) electromyographic instrument with a wireless telemetry system. The sampling rate will be 1500Hz with a 10Hz high-band digital filter and 500Hz low-band pass. Root mean square (RMS) values will be calculated and used as the variables studied for the electromyographic activity outcome (secondary outcome). EMG activity measurements will only be taken at two time points: baseline and day 5.
The electrode sites will be prepared by trichotomy (hair scraping) in the vicinity of the muscle belly, followed by skin cleansing using isopropyl alcohol with a sterile gauze to reduce impedance to the EMG signal. Disposable Ag/AgCl surface electrodes will then be fixed in the appropriate places [22, 23].
The electrodes will be positioned on the distal portion of the quadriceps muscle, superficially to the vastus lateralis and the vastus medialis. Specifically, the electrodes over the vastus lateralis will be placed 2/3 of the line between the anterior iliac spine superior to the lateral side of the patella, while the electrodes over the vastus medialis will be placed at 80% of the line between the anterior iliac spine and the joint space in front of the anterior edge of the medial ligament, as recommended by SENIAM (Surface Electromyography for the Non-Invasive Assessment of Muscles).
In both muscles, two electrodes will be placed approximately 20 mm apart, in the direction of muscle fibers. The pairs of electrodes will be placed parallel to the target muscle fibers.
During horizontal jumping, we will measure the activities of the vastus medial and lateral muscles. To allow for comparisons of muscle activation within and between participants, we will need to normalize the electromyography (EMG) signals [24]. For our study, we will use the maximum voluntary isometric contraction (MVIC) method, which is commonly used to normalize EMG signals [25]. Specifically, we will perform three MVICs, each lasting three seconds with a one-minute rest period between each repetition [26].
To obtain MVIC values for the vastus lateralis and vastus medial muscles, participants should assume the sitting position with the hip and knee at 90° flexion. Individuals will be instructed to maintain an upright trunk position with their arms crossed over the chest [27]. The adjustable nylon strap will be positioned on the distal third of the leg, near the ankle, to apply resistance during knee extension movement.
Subjects will be instructed to maintain maximum voluntary isometric contraction over the three seconds against resistance. A practice attempt will be made before the 3 attempts collected, to ensure the proper completion of the tasks. Verbal encouragement will be provided during all attempts [27].
Treatment arms
Following the participants' assessments, they will be randomly allocated into two groups: the Intervention Group (IG) and the Control Group (CG) (Fig. 01). An independent researcher (Researcher 02) will use the research randomizer software to generate a coded sequence for randomization. This randomized sequence will be placed in a numbered opaque envelope, and another independent researcher (Researcher 03) will use it to allocate participants to their respective groups.
The double-blinding process will be achieved by blinding the evaluator (Researcher 01) and the therapist (Researcher 04). The blind assessor will not know whether the participant belongs to the intervention or control group. The therapist will be blinded to whether they will be applying the laser with active parameters or not [28].
Control group
Participants allocated to the control group will receive placebo laser therapy. The researcher (Researcher 03) responsible for adjusting the laser will ensure that it is set to placebo parameters, where the dosage will be zeroed. The device will emit the same sounds regardless of the programmed mode (active laser or placebo), and the infrared light will not be visible [29]. The therapist (Researcher 04) who will apply the laser, whether active or placebo, will not be aware of the equipment's configurations.
Intervention group
The independent researcher (Researcher 03) will know that the application of laser therapy (laser recover; MMO, Brazil) in this group should be active. In this study, the application procedure will involve stationary contact with the skin, using a wavelength of 808nm (infrared), a power of 100mW, and the number of points applied will vary according to the size of the participants' muscles, ranging from 5 to 8 points for vastus medialis and 10 to 15 points for vastus lateralis. The firing time will be determined by the dosage established in the equipment, which will be 60 seconds per point. The dosage of 40 J will be divided by the number of points in J/cm², and the size of the point on the skin will be 3mm², referring to the output area of the equipment and spot area of 0.03cm².
Sample Calculation
The sample size calculation was performed using the GPower v.3.1 program [30]. Based on the nature of the study, a statistical design of f-test of repeated measurements and interaction between-within factors, with 5 repeated measurements, and two groups studied, a statistical power of 0.80, an alpha of 0.05, and an effect size of 0.25, the sample size was calculated to be 20 individuals. To account for potential sample loss (20%), which is common in clinical trials, the total sample size will be 24 participants allocated to both groups.
Statistical Analysis
The statistical analysis inferential will be made by intention of treatment. After confirmation of normality (Shapiro-Wilk test), homoscedasticity (Levene test) and imputation of means for missing data of variables with normal distribution, ANOVA 2 factors will be performed for repeated measures, followed by Newman-Keuls post-test, to obtain the group effect (intervention and control), time effect (Day 01, 02, 03, 04 and 05) interaction effect (group x time).
Significant differences will be considered with a α of 5%, but for the description of the effect of the intervention will also be calculated the size of the effect (Cohen's coefficient d) and the difference between the means and their respective 95% confidence interval.
Table 1: Procedures that will be carried out over the 5 days of study.
