Study design
This is a simple blind placebo controlled clinical trial conducted at the Center for Studies and Care in Physiotherapy and Rehabilitation at Universidade Estadual Paulista (FCT/UNESP), Presidente Prudente, SP, Brazil. The trial was registered at ClinicalTrials.gov (NCT04420819) and approved by the Research Ethics Committee of FCT / UNESP, Presidente Prudente, SP, Brazil (CAAE: 30765020.3.0000.5402).
The study protocol follows the SPIRIT 2013 checklist (Standard Protocol Items: Recommendations for International Trials) (25) (Supplementary File 1) and the TIDieR (Template for Intervention Description and Replication) (26), so that the information and quality of reports of interventions are well described (27).
Participants and population analysis
For this study, the sample will consist of 80 healthy male individuals classified as active by the International Physical Activity Questionnaire (IPAQ) (28), aged between 18 and 35 years, who will be recruited from a database of the Sports Physiotherapy Laboratory of FCT/UNESP and the community, through dissemination folders and posters on the institution's premises, social networks, and advertisements in the local media. These procedures are recommended by Treweek et al.(29) as strategies to improve participant recruitment.
Individuals who exhibit one or more of the following characteristics will not be included: (1) the presence of any health condition that contraindicates or prevents EE; (2) diabetes and diagnosed arterial hypertension; (3) inflammatory, psychiatric, cardiovascular, and/or respiratory rheumatological disease; (4) being an alcoholic, using drugs, and/or being a smoker; (5) history of knee surgery (for example, meniscal repair and ligament reconstruction) or recent musculoskeletal injury to the lower limbs that may impair performance during tests or interventions (for example, muscle injury, tendinopathy, patellofemoral pain in the lower limbs, and/or back pain in the previous six months); (6) involvement in any type of training program during the study period; (7) engaging in a lower limb strength training program during the three months prior to participating in the study; (8) use of ergogenic supplements to improve physical performance and/or muscle mass and/or vasoactive drugs; (9) having one or more risk factors predisposing to thromboembolism (30).
Participants will be excluded from the study if they: present any health problem that does not allow continuity, use medication, electrotherapy or other therapeutic means that may interfere with any result, perform unusual or strenuous physical activities during the evaluation period, or wish to leave the study.
Participants will be instructed in advance not to perform any physical activity or to use any therapeutic form of pain relief or performance improvement during the collection. They will also be instructed not to consume alcohol during the procedure. If there are episodes of musculoskeletal injuries, individualized physiotherapeutic treatment will be offered.
The flowchart of the study design and the composition of the groups is illustrated in figure 1.
Randomization
The randomization sequence will be developed by a researcher who will not be involved in recruiting, evaluating, or training participants using software (Microsoft Office Excel 2007) and will be placed in sequential numbering in opaque and sealed envelopes. There will be blinding of the evaluators and statistical analysis procedures. The experimental protocol will be supervised by trained physiotherapists who are not involved in the randomization process or assessments. Due to the nature of the interventions, the participants who perform the exercise will not be blind to the allocation of the groups.
Participants who meet the eligibility criteria will be randomly allocated in a 1: 1: 1: 1 ratio to one of the four groups, namely:
- IPC-TOP: this group will carry out baseline assessments, perform IPC using exactly the TOP, then perform post-IPC assessments and start EE. Post-exercise assessments will take place immediately after the end of EE and will be repeated at 24h, 48h, 72h, and 96h.
- IPC-40%: this group will perform baseline assessments, perform IPC using 40% more occlusion than TOP, then perform post-IPC assessments and start EE. Post-exercise assessments will take place immediately after completion of the EE and will be repeated at 24h, 48h, 72h, and 96h.
- IPC-10mmHg: this group will carry out baseline assessments, perform the occlusion-perfusion intervention with 10mmHg restriction characterizing the placebo, and then perform post-IPC assessments and start EE. Post-exercise assessments will take place immediately after completion of the EE and will be repeated at 24h, 48h, 72h, and 96h.
- CONTR: this group will carry out the baseline assessments, and immediately afterwards start the EE. Post-exercise assessments will take place immediately after the end of the EE and will be repeated at 24h, 48h, 72h, and 96h.
Study design
Data collection will be carried out at the Center for Studies and Assistance in Physiotherapy and Rehabilitation (CEAFIR) of FCT/UNESP, respecting the time from 17h to 22h. All procedures will be performed under standard conditions (temperature: 21-23°C; relative humidity: 40-60%). Each participant will attend the clinic for five consecutive days. Initially, participants will be assessed for anthropometric characteristics, using a scale (Tanita BC 554, Iron Man/Inner, Arlington Heights Illinois, USA) and a stadiometer (Sany - American Medical do Brasil, São Paulo, Brazil) from which the body mass index (BMI) will be calculated.
After these initial procedures, the TOP evaluation will be carried out. After a 10-minute rest, baseline outcome assessments will be performed. Initially, CK and blood lactate will be collected, followed by application of the scales of perception of recovery and muscle pain and the pain threshold. In the supine position, the participant will rest for 10 minutes before the ultrasound, myotonometry, and bioelectrical impedance (BIA) evaluations are performed. Subsequently, MVIC will be evaluated. Next, the participants will perform the previously randomized 40-minute IPC protocol. Evaluations will be carried out immediately after the IPC protocol of all analyzed outcomes except for ultrasound. The same order of execution of the evaluations will be maintained. Thereafter, EE will be initiated and immediately after the end of the EE, all outcomes will be collected again. Subsequent visits will be carried out 24, 48, 72, and 96 hours after the EE, in which the same outcomes mentioned above will be collected, maintaining the same order of execution of the evaluations. The study design is outlined in figure 2.
TOP determination
After assessing the anthropometric parameters, the participants will be asked to lie down for ten minutes (31). All participants will be instructed to avoid strenuous exercise and alcohol intake within 48 hours of the TOP assessment.
For the determination of TOP, a transducer with Doppler equipment (DV-2001; Medpej, Ribeirão Preto, São Paulo, Brazil) will be used, which will be positioned over the posterior tibial artery to capture the auscultatory pulse located at the average distance between the medial malleolus tibia and Achilles tendon. A blood pressure cuff will be attached to the participant's thigh close to the region of the inguinal crease of the dominant member (30) and will then be inflated to the point where the auscultatory pulse of the tibial artery is interrupted. TOP will be defined as the pressure at the moment when the arterial pulse is abolished, indicated by the absence of an auscultatory signal.
The vascular occlusion will be performed using an adapted blood pressure cuff (nylon, velcro, 175 mm wide and 920 mm long, JPJ - Hospital Materials Industry, São Paulo, Brazil). We opted for a wider cuff, as it has been proven that the width of the cuff has great influence on the pressure required to achieve total blood flow restriction (32).
IPC Protocol
The IPC protocol will be applied to the inguinal region of the dominant limb with the participants relaxed and comfortably positioned in the supine position. The same cuff used to determine TOP will be used and the protocol will consist of four cycles of total ischemia (TOP determined individually) of five minutes, followed immediately by four cycles of five minutes of vascular reperfusion (0 mmHg), totaling 40 minutes, as shown in figure 3.
To perform the IPC protocol, one of the study groups will use 40% more than the TOP, according to the study by Lopes et al. (33), the pressure at which the blood flow stops passing to the tibial artery varies from 140 to 160 mmHg and most studies performing IPC use pressures between 200 to 220 mmHg, thus, the values are corresponding (33). The other study group will use the exact TOP, since at this value there is an absence of blood flow.
Placebo preconditioning
Placebo preconditioning will be performed on the dominant thigh with the same cuff, similar to the IPC protocol described above (Figure 3), but with four cycles of five minutes of placebo occlusion (10mmHg), alternating with four cycles of five minutes of reperfusion (0 mmHg) (34, 35).
It should be mentioned that in the three study groups, participants will be previously informed that the applied occlusion pressure will be sufficient to improve performance and avoid muscle damage. In addition, all procedures will be performed individually, to prevent participants from talking to each other about the compression generated by the cuff.
EE Protocol
The EE will be performed on an isokinetic dynamometer (Biodex System 4 Pro, New York, New York, USA) for the knee extensor muscles of the dominant limb. Initially, five submaximal knee extension contractions will be performed for familiarization. Before each repetition, the dominant leg will be positioned at 30º of knee flexion. The participant will be instructed to perform a knee extension, while the dynamometer, with its resistance, returns the leg to 90º flexion, at a speed of 60º/s (1.04 rad/s) executing a range of movement of 60º (30-90º of knee flexion).
The protocol is based on the study by Machado et al. (22), starting five minutes after familiarization and consisting of 5 sets of 15 maximum eccentric contractions of knee extension, with 30 seconds of rest between sets, totaling 75 repetitions. The speed and range of movement will be similar to the familiarization and verbal encouragement will be given throughout the protocol. According to the authors, this protocol is capable of promoting muscle damage (22).
Primary and secondary outcomes and assessment points
Primary outcomes will be related to muscle damage and muscle function through the quantification of creatine kinase, blood lactate, MVIC, and muscle thickness. In addition, there will be four measures of secondary outcomes: assessment of pain and pain threshold, assessment of recovery perception, myotonometry (muscle tone, stiffness, and elasticity), and electrical bioimpedance (resistance, reactance, and phase angle). All outcomes will be collected at the initial assessment with the exception of the perception of recovery, and immediately and 24, 48, 72, and 96 hours after the end of the EE. Ultrasonography will not be performed immediately after the exercise. The description of the specific moments of collection of outcomes can be seen in Table 1.
Table 1. Time points for outcomes
|
Outcomes
|
Initial assessment
|
Immediately after exercise
|
Recovery
|
24 hours
|
48 hours
|
72 hours
|
96 hours
|
Primary
|
Muscle damage
|
CK
|
√
|
√
|
√
|
√
|
√
|
√
|
Blood lactate
|
[Lact+]
|
√
|
√
|
√
|
√
|
√
|
√
|
Muscle strength
|
MVIC
|
√
|
√
|
√
|
√
|
√
|
√
|
Muscle thickness
|
Ultrasound
|
√
|
|
√
|
√
|
√
|
√
|
Secondary
|
Pain
|
VAS
|
√
|
√
|
√
|
√
|
√
|
√
|
Pain threshold
|
Algometer
|
√
|
√
|
√
|
√
|
√
|
√
|
Perception of recovery
|
Likert scale
|
√
|
√
|
√
|
√
|
√
|
√
|
Muscle tone, stiffness and elasticity
|
Myotometry
|
√
|
√
|
√
|
√
|
√
|
√
|
Resistance, reactance and phase angle
|
Electrical bioimpedance
|
√
|
√
|
√
|
√
|
√
|
√
|
Details of procedures
Creatine Kinase (CK)
Plasma CK concentration will be obtained by means of 32 μL of capillary blood collected from the digital pulp. This puncture will take place by means of a lancet with automatic trigger, after cleaning the location with 95% ethyl alcohol and drying with cotton. The blood sample will be drained into a heparinized capillary tube and then pipetted into a reactive CK strip for analysis on the Reflotron Plus System (Roche Diagnostics, Mannheim, Germany) using the reflection photometry method at 37 ° C (test temperature). The test strips will be kept at a storage temperature of 2 to 8 ° C, according to the manufacturer's instructions. The Reflotron System method allows fast and reliable measurement of CK levels (36).
Blood lactate concentration
To assess the participant's blood lactate concentration, 25 ml of blood will be collected from an ear lobe capillary. Heparinized capillaries and polyethylene Eppendorf tubes (1.5 ml) containing 50 μL of sodium fluoride (NaF - 1%) will be used. The analyses will be performed on a lactimeter (YSI, Yellow Springs - 1,500) (37) and the lactate values will be expressed in mmol/L. The lactate will be collected at the following moments: pre IPC protocol, pre EE, immediately after the end of EE, and in the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, and 15th minutes, and 24 h, 48 h, 72 h, and 96 h after the intervention.
Perception of recovery
The perception of recovery of the lower limb submitted to the EE protocol will be assessed using a 10-point Likert Scale, where 1 indicates “not recovered” and 10 “fully recovered” (22). The scale will be presented to the participants and, so that they are not influenced by the researcher, they will answer the question: "From 1 to 10 points, how do you rate the perception of recovery felt in your lower limb at this moment?" (22,382).This scale will be applied immediately after the end of the EE, and 24, 48, 72, and 96 hours after the intervention.
Muscle pain and pain threshold
Muscle pain will be measured using the visual analog scale (VAS). Participants will be asked to rate their exercise-induced leg pain, on the scale, which ranges from 0 “no pain” to 10 “extreme pain” (39).
To assess the pain threshold, a pressure algometer will be used, which is a reliable and validated instrument (FPX 50/220; Wagner instruments, Greenwich, Connecticut, USA) (40). The pressure algometer will be applied 4 cm above the base of the patella, 15 cm below the antero-superior iliac spine (ASIS), midpoint between the patella and the antero-inferior iliac spine (AIIS), and 2 cm medial and 2 cm lateral in relation to the midpoint. The pain threshold will be defined in kgf and will not exceed 2.55 kgf, as suggested by Jӧnhagen et al. (41).
Muscle thickness
The evaluation of the muscular structure will be carried out using ultrasound images of the participant's dominant lower limb, which will be captured using Siemens Sonoline Sienna equipment (Issaquah, WA, USA), together with a linear matrix transducer (48 mm, 7, 5 MHz) to determine the thickness of the rectus femoris (RF) and vastus lateralis (VL) muscles. Anatomical reference points and skin marks will be drawn on transparent sheets to ensure similar positioning of the ultrasound transducer in the same location in the evaluations.
The images will be taken between the midpoint of the greater trochanter and the lateral condyle of the femur (42). The ultrasound transducer will be covered with water-soluble transmission gel and positioned perpendicular to the skin over the RF and VL and oriented parallel to the muscle fascicles (42). The alignment of the transducer will be considered adequate when several fascicles can be drawn without interruption through the image (42).
Myotonometry
MyotonPRO (MyotonAS, Tallinn, Estonia) will be used to measure tonus, stiffness, and elasticity of the quadriceps femoris muscle (43). The device will be positioned at 2/3 between the ASIS and the upper pole of the patella (44), at the midpoint between them and 2 cm to the medial and 2 cm to the side, 15 cm from the AIIS and 4 cm from the base of the patella.
The device will be positioned perpendicular to the evaluated region, with light pressure. This preload is controlled and corresponds to 0.18 N of initial compression of the subcutaneous tissue, after which an additional impulse of 0.40 N will be released, with a duration of 15 ms. This impulse will induce oscillation in the tissue, which will be damped or deteriorated (45).
BIA
BIA will be assessed using tetrapolar electrodes (BIA Analyzer, Nutritional Solutions Corporation, Harrisville, MI, USA, f = 50 kHz, and 800µA) (46, 47). Global and localized evaluations will be carried out. The global assessment will take place with the participant in the supine position; the electrodes will be positioned on the dominant hand (base of the 3rd metacarpal phalanx and between the styloid process of the radius and the head of the ulna) and on the dominant foot (at the base of the 3rd metacarpal phalanx and in the anterior region of the ankle, between the malleoli). The localized evaluation will be carried out to evaluate the quadriceps femoris muscles; the electrodes will be positioned five centimeters below the EIAS and above the base of the patella. The BIA components analyzed will be resistance R, reactance (Xc), and phase angle (phA) (47, 48). The analysis of the tolerance ellipse will also be performed. The Bioscan program: BL-960141 (Biologica, Barcelona, Spain) will be used for the analyses (46, 47).
MVIC
For MVIC evaluation, the participant will be positioned with the dominant lower limb on the Biodex System Pro isokinetic dynamometer (Biodex Medical System, Shirley – NY, USA). According to the protocol suggested by Baroni et al. (49), prior to the evaluation, the volunteer will be submitted to a warm-up, which will consist of ten repetitions of concentric contractions of knee flexion-extension at 180º/s throughout the range of motion.
Muscle function will be assessed by means of the highest torque value obtained between three repetitions of five seconds of maximum voluntary contraction at 60º of knee flexion (with 0º corresponding to the maximum extension). A two-minute interval between repetitions will be administered in order to minimize the possible effects of fatigue. The participant will be instructed to give their maximum strength and will be verbally encouraged by the researcher in each maximum voluntary contraction.
Sample calculation
The sample calculation was performed based on the study by Bailey et al.(50), using the CK variable, since this variable expresses the amount of muscle protein in the blood representative of indirect muscle damage. Considering the standard deviation in the CK concentration of 200 μL, using a two-tailed hypothesis test with 80% test power and 5% significance level, a sample size of 20 participants per group was stipulated, totaling 80 participants.
Statistical analysis
The normality of the data will be verified by the Kolmogorov Smirnov test. If normality is detected, the sample characterization variables will be presented as mean and standard deviation, if not, as median and interquartile range. To compare the characterization of the sample, one-way ANOVA with Tukey's post-test or Kruskal-Wallis with Dunn's post-test will be used depending on the normality of the data.
The comparisons of the outcomes between the four groups studied and the moments will be performed using the technique of analysis of variance for repeated measures model in the two-factor scheme, which will provide information on the effects of time, group, and interaction. The repeated measurement data will be checked for sphericity violation using the Mauchly’s test and the Greenhouse-Geisser correction will be used when the sphericity is violated. For moment analysis, Bonferroni's post-test for parametric distribution or Dunn's post-test for non-parametric distribution will be used and the analysis between the groups will be performed using One-Way ANOVA or the Kruskal Wallis test.
In addition, the effects of the groups studied will be verified for all the outcomes assessed by calculating the effect size (ES) using Cohen's d, considered as “null” (<0.2), “small” (≥0.2), “moderate” (≥0.6), “large” (≥1.2), or “very large” (≥2.0) (51).
An intention-to-treat analysis will be performed using the patient’s most recent assessment in case of withdrawals or absence of data. The level of significance will be p <0.05 for all tests. The statistical program SPSS (version 24.0) (SPSS Inc., Chicago, IL, USA) will be used for the analyses.