Aim
The aim of this study is to propose a new methodology to determine the IMT load for recreational cyclists, which promotes better results for physical performance and for cardiovascular, respiratory and metabolic responses compared to traditional methodologies.
Primary Outcome Measures:
The performance in exercise: peak oxygen uptake (VO2PEAK) and work load (W) were obtained by cardiopulmonary exercise testing (CPET).
Secondary Outcome Measures:
· The cardiovascular responses to IMT: systolic arterial pressure (SAP), dyastolic arterial pressure (DAP), heart rate (HR), cardiac output (CO), stroke volume (SV) and total peripheral vascular resistance (PVR) will be evaluated during the CPET and incremental respiratory muscle endurance test (RMEi);
· The respiratory responses to IMT: carbon dioxide production (VCO2), respiratory exchange rate (RER), respiratory rate (RR), respiratory ventilation (VE), oxygen uptake efficiency slope (OUES) and minute ventilation-carbon dioxide production slope (VE/VCO2SLOPE) will be monitored and registered during CPET and RMEi. MIP and MEP will be collected in the RMS test. The pulmonary function test (PFT) index comprises: slow and forced vital capacity (SVC and FVC), maximal voluntary ventilation (MVV), forced expiratory volume in 1 second (FEV1), relationship between FEV1 and CVF (FEV1/FVC), IC, expiratory reserve volume (ERV) and MVV;
· The metabolic responses to IMT: oxyhemoglobin (O2Hb), deoxyhemoglobin (HHb) and total hemoglobin (tHb) and VO2PEAK responses will be measured during CPET and RMEi;
Study design
This is an experimental, longitudinal, randomized, controlled and double-blind study. The research participants and the researcher, who will perform the evaluation and reevaluation protocols and statistical analysis (PRS), will be blinded. The researcher who will carry out the analyses of the study (tabulation, data processing and statistical analyses) will not participate in the randomization and training of the participants. Thus, after tabulating all the data, the same person (PRS) will receive a table with the data from the other researchers, separated in the training groups, using codes to perform the statistical analysis. For the blindness of the participants, a barrier will be fixed on the computer screen, which will prevent them from accompanying the training loads, which are being submitted. The methodological design was based on the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) (30).
Subjects
GPower software 3.1.3 was used to calculate the sample size required for this study. To determine the sample size, the mean effect size value [f = 0.25, according to COHEN(31)] was used for the two-way mixed ANOVA test, values of 0.05 for the type I probability error (α), of 0.80 for the type II probability error (power or β). Therefore, in order to ensure these pre-established conditions, at least 30 subjects should participate in this study.
Thirty recreational male cyclists, aged between 20 and 40 years, will be evaluated. They will be randomly divided into three groups: SHAM group (SG), PThC group (CPG) and 60% of the MIP group (60G). The groups will be formed by the randomization of the paired individuals with the same aerobic functional classification (32) and age group (subdivided into decades), using R software 3.0.2 to allocate the subjects.
The criteria used to select the participants will be: male subjects aged 20 to 40 years old, apparently healthy, who have been practicing cycling for at least 6 consecutive months and at least 150 minutes/week [classified as active by the AMERICAN COLLEGE OF SPORTS MEDICINE (33)], non-smokers, non-alcoholics, or users of illicit drugs or medication that may interfere with the results of the research. In addition, participants will not be able to present abnormalities in the cardiovascular, respiratory, orthopedic or neurological systems, nor of other systems that make the proposed tests impossible. The exclusion criteria of the study is as follows: participants with electrocardiogram (ECG) changes [ischemia, overload, severe arrhythmias (such as ventricular tachycardia) and conduction disorders], both at rest and during the clinical exercise test, obese [body mass index (BMI) >30 kg/m²], diabetics, hypertensive, participants with alterations in laboratory tests, participants with respiratory muscle weakness [MIP<60% predicted (34)], former smokers with at least 1 year of interruption, those who are illiterate and/or who do not have a sufficient level of understanding to understand the routine of the protocols, as well and participants who have performed some type of IMT over the last 12 months.
The participants will be recruited by electronic and printed media, as well as contacts with subjects that are part of the database from the Laboratory of Cardiovascular Physiotherapy (LFCV) at the Federal University of São Carlos (UFSCar). After identifying the eligible participants, they will be invited to participate in the study and after they have accepted, they will carry out all the evaluations described below, as well as the type of training to which they are allocated.
Study planning
The present study will be conducted in accordance with the Helsinki Declaration. This study was approved by the Human Research Ethics Committee at the Federal University of São Carlos (UFSCar) (nº 1.558.731). All subjects signed an informed consent form.
The experimental tests and procedures will be performed at the LFCV at the Nucleus of Research in Physical Exercise (NUPEF) at the Department of Physical Therapy (DFisio) and the clinical ergometric tests at the Cardiovascular Physiotherapy area of the School Health Unit (USE), both located at UFSCar, São Carlos campus. The blood tests will be performed at the Clinical Analysis Laboratory at UNIMED (a cooperative medical system) in São Carlos (UNILAB).
Environmental conditions will be controlled, and participants will be instructed according to Perseguini et al. (35). The tests will always be performed in the afternoon, considering the influence of the circadian cycle on the evaluation results.
On the day of the tests, previously to applying the experimental protocols, the conditions related to the participants´ state of health will be observed. Moreover, prior to the tests, participants will be familiarized with the equipment, respiratory maneuvers and the subjective perception of effort scale – BORG/CR10 (36) in order to reduce participants' anxiety and prevent the effect of the learning process from affecting the search results.
Clinical evaluation and characterization of the sample
Before performing the experimental protocols, the participants will be submitted to the following evaluations: anamnesis, conventional 12-lead ECG, treadmill clinical exercise test, both performed in the presence of a cardiologist, assisted by a physiotherapist. The aim of these tests will be the clinical and cardiovascular evaluation of the participants.
To characterize the participants and verify their health status, the following evaluations will be performed before the experimental protocol: body composition evaluation by Dual-energy X-ray Absorptiometry (DXA)(Discovery DXA System, Hologic, USA), nutritional assessment and blood tests (lipid profile, total cholesterol, High density lipoproteins (HDL), Low density lipoproteins (LDL), urea, creatinine, fasting glucose, uric acid, insulin resistance, C-reative protein and glycated hemoglobin to check their health status.
Experimental Protocol
The experimental protocol will take place over thirteen weeks, as shown in Figure 1. In the first, fifth, ninth and thirteenth weeks, the participants will be submitted to the following evaluations: CPET, RMS, iRME and constant load test (CLT). The pulmonary function test (PFT) will be performed only in the first and thirteenth weeks of the study. The IMT will be carried out for 11 weeks. It is emphasized that during the reevaluation weeks, the participants will continue doing the training.
The aim of the re-evaluations, which will be carried out in the fifth and ninth weeks of training, is to readjust the training loads, as well as follow the responses to the IMT.
Respiratory muscle strength tests (RMS)
The evaluation of RMS will be performed with the volunteers at rest in the sitting position, using a digital manovacuometer (MVD-300, Globalmed, Porto Alegre, Brazil) and a nasal clip, according to the Brazilian Guidelines for measuring maximal static respiratory pressures (37). This measure will always be carried out by the same appraiser.
MIP will be determined after maximal inspiratory effort, from the residual volume. The maximal expiratory pressure (MEP) will be determined after maximal expiratory effort, from total lung capacity. These maneuvers will be performed against a rigid tube, occluded distally and a 2 mm-hole mouthpiece (37) will be used. The values of the maximal respiratory pressures will be those observed in the first second after the peak of pressure (37). At least three maneuvers will be performed, with a 30s interval between each maneuver (38), obtaining the highest reproducible values (difference <10%) found in at least three maneuvers and considering the maximal respiratory pressure value as the highest value. Normal values will be based on the regression equation proposed by Neder et al. (39) for the Brazilian population. Respiratory muscle weakness, maximal static pressure values <60% of those predicted, will be considered (34).
Cardiopulmonary Test (CPET)
The CPET will be used to assess the aerobic power of the participants [peak oxygen uptake (VO₂PEAK)] (40) to determine the gas exchange threshold (GET) by the ventilatory method (41), the respiratory compensation point (RCP) (40) and inspiratory muscle metaboreflex (42).
A ramp type protocol will be performed on an electromagnetic braking cycle ergometer (CORIVAL V3, Lode BV, The Netherlands) and will consist of a 6-minute rest, 3-minute free-load warm up and a gradual increase in load until the exercise is stopped, followed by 6 min active recovery and 1 min passive recovery. The power increment will be calculated for each participant according to the values established by the formula described by Wasserman et al. (43) and adapted according to the evaluator's experience, preventing the increment from being underestimated.
Participants will be instructed to maintain a cadence between 60 and 80 rpm throughout the protocol and the test will last from 8 to 12 minutes (43). Three independent evaluators will determine the GET and the RCP. The highest value of VO2 obtained in the last 30 seconds of the CPET will be considered the VO2PEAK (40). In addition, the following variables will be evaluated in the GET and peak effort: VO2, carbon dioxide production (VCO2), pulmonary ventilation (VE), oxygen uptake efficiency slope (OUES) and minute ventilation-carbon dioxide production slope (VE/VCO2 slope) (40,44).
The activation of the inspiratory muscle metaborreflex will be evaluated by analyzing the behavior of the variables: oxyhemoglobin (O2Hb), deoxyhemoglobin (HHb) and total hemoglobin (tHb) obtained by Near Infrared Spectroscopy (NIRS) and the cardiovascular data, heart rate (HR) and mean arterial pressure (MAP), obtained by a bioamplifier for ECG signals (BioAmp FE132) and a Finometer (Finapres Medical Systems, The Netherlands), respectively, at the intensities of 50% to 100% of the VO2PEAK, subdivided into 10% intervals. In addition, the activation point of the inspiratory muscle metaboreflex will be considered the moment at which the nonlinear HR and MAP, as well as oxygenation decrease for the vastus lateralis (VL) and increase for the external intercostal (EI), i.e. redirection of blood flow from the peripheral musculature to the respiratory muscles (6).
Pulmonary function test (PFT)
This examination will be performed according to the international standard (45) and will consist of tests of slow and forced vital capacity (SVC,and FVC) and maximal voluntary ventilation (MVV). The test will be performed using a flow module coupled with a system of ventilatory and metabolic measurements (ULTIMA MedGraphics - St. Paul, Minnesota, USA). The analyzed variables will be: SVC, FVC, forced expiratory volume in 1 second (FEV1), relationship between FEV1 and CVF (FEV1/FVC), IC, expiratory reserve volume (ERV) and MVV. The predicted values will be calculated according to Pereira (46).
Protocol for the determination of critical inspiratory pressure (PThC)
An incremental protocol with a load of 50 to 100% MIP (Figure 2) will be performed and 10% of MIP will be added every 3 min. The participant will receive a verbal stimulus to maintain the respiratory rate (RR) in 12 breaths/minute and the test may continue until he reaches 100% of MIP. In this case, if the participant can generate an air flow capable of triggering the equipment more than once in this load, the MIP measurement will be redone and the test repeated. The following will be considered: failure to maintain the stipulated load at 12 breaths/minute for at least 1 minute, failure to maintain respiratory effort indicated by the volunteer (BORG/CR10≥7) (36) and the participant requesting interruption. The highest percentage of MIP that the volunteer is able to maintain for at least 1 min (PThMÁX) will be stipulated as the RME measurement (26–28,47).
After determining the PThMÁX, individuals will carry out incursions against a constant load, without the RR being controlled in order to identify the total time tolerated in each load (TLIM). Individuals will be exposed to three different resistances (95%, 100% and 105% PThMÁX) with a 15-minute interval between them. The order of resistances will be determined by holding a draw without the individual knowing the order of the charges in which he will be subjected to.
After obtaining the TLIM of each load, a pressure graph will be plotted by time and the PThC will be determined by using a linear regression between the variables (23).
Monitoring the experimental protocol
The equipment described below will be used to monitor the participants in the evaluations of the CPET and iRME.
Metabolic and respiratory variables
The ventilatory and metabolic variables will be collected, breath by breath, through a system of expired gas measurements (ULTIMA MedGraphics - St. Paul, Minnesota, USA) and processed through specific software (Breeze Suite 7.1, MedGraphics - St. Paul, Minnesota, USA). In addition, the BORG/CR10 scale (36) will be used to assess the subjective perception of the exercise performed by the participant.
Cardiovascular variables
The acquisition of the ECG and arterial pressure (AP) signals to evaluate the cardiovascular responses will be performed at a sampling frequency of 1.000 Hz. The ECG signals will be captured by means of the CM5 lead. The HR will be recorded and stored beat-to-beat. Electrocardiographic signals will be captured and processed via an interface between a bioamplifier for ECG signals (BioAmp FE132, ADInstruments, Australia) and a biological signal acquisition system (Power Lab 8/35, ADInstruments, Australia) and a microcomputer (Intel I5).
On the other hand, pulse pressure will be captured using Finometer Pro® (Finapres Medical Systems, The Netherlands), which allows non-invasive measurements of pulse arterial pressure (FinAP), beat-to-beat, obtained by positioning a cuff in the third phalanx of the third finger of the left hand. The equipment will be calibrated according to the manufacturer's instructions. In addition to pulse AP, the values of cardiac output (CO), stroke volume (SV) and total peripheral vascular resistance (PVR), derived from the AP curves and analyzed in the Beat Scope® Easy software (Finapress Medical Systems, The Netherlands) will be evaluated.
Metabolic evaluation by NIRS
The EI and VL muscle oxygenation variables will be measured by the NIRS (Oxymon Mk III, Artinis Medical Systems, The Netherlands). Two optodes will be used, which will be positioned as described below: IE = eighth left intercostal space in the anterior axillary line; VL = 12 to 15 cm of the knee joint, lateral line between the greater trochanter of the femur and the patella (48).
The sampling rate of the device will be set at 250 Hz. Inter-ops distance will be 35 mm for the EI and 40 for the VL (48). The differential path-length factor (DPF) will be 4 for the EI muscles and 3.83 for the VL. The calibration will be redone for each subject and the data will be continuously captured during the CPET and the iRME. For each muscle, the change in tissue oxygenation and local blood volume will be estimated by changes in O2Hb, HHb and tHb, calculated automatically by the software of the equipment by the formula (tHB = O2Hb + HHb).
Inspiratory muscle training (IMT)
Training Description
The training will last for 11 weeks, with a weekly frequency of 3 sessions and each session will last one hour each. Each session will consist of a 5-minute warm-up, where each volunteer will perform a constant loading protocol with 50% of their training load. The training protocol will consist of 3 sets of 15 minutes of breaths, with a 1-minute interval between them (Figure 3).
The loads that will be used in the training will be: for the CPG the value of the PThC, for the 60G, the resistance will be 60% of MIP and for the SG, the training will be carried out with a resistance of 6 cmH2O. All groups will perform IMT using the Linear Load Respiratory (PowerBreathe inspiratory muscle trainer, Ironman K5, HaB Ltd, UK).
The volunteers will be instructed to maintain diaphragmatic breathing and RR of 12 breaths/minute for the entire duration of the training protocol, and the RR will be controlled by using a recorded verbal command, ensuring that all participants receive the same stimulus. During the 11 weeks of training, volunteers will be instructed not to change their activities and physical training or food intake.
Every day, before and after the training protocol, the AP will be checked, and the health status of the volunteers will be observed while the HR will be monitored, recorded and stored during all training sessions by a Polar 800CX (Kempele, Finland).
In addition, during the training period, the participants will be asked to complete a physical activity schedule to monitor the activities performed by each individual during the research participation and complete a food survey that will be analyzed later by a nutritionist, thus avoiding these factors interfering with training responses. The weekly training volume and the end, which will be controlled over 11 weeks, will be ensured for all participants throughout this study.
Volunteers who do not complete the 3 weekly training sessions and/or the 33 total training sessions, or participants who change their physical activities, their eating habits, or those who begin to use any supplement or medication continuously will be excluded during the survey.
Statistical analysis
Due to the lack of studies that evaluate the effect of different loads of IMT, among them PThC, on the physical performance in recreational cyclists, we chose to carry out the statistical analysis considering the size of effect desired by the study, as proposed by Cohen (31). Thus, the sample calculation was performed in the GPower software 3.1.3 to determine the sample size, the mean effect size value [f = 0.25, according to COHEN (31)] was used for the two-way mixed ANOVA test, values of 0.05 for the type I probability error (α), of 0.80 for the type II probability error (power or β). Therefore, in order to ensure these pre-established conditions, at least 30 subjects should participate in this study.
All statistical analyses will be processed using the SPSS Statistics software 17.0 (SPSS Inc., Chicago, Ill., US). The level of significance will be set at p <0.05. Data with normal distribution will be presented according to the mean±standard deviation; and those with non-normal distribution, according to median (interquartile range).
The Shapiro-Wilk test will be used for data analysis normality testing and the Levene test for the homogeneity evaluation. A descriptive analysis of the three groups evaluated and then the paired t-test will be performed on the following data: age, anthropometric evaluation, body composition densitometric analysis and blood test results.
To analyze the comparison between the three groups, considering the following factors: group and training time, in the variables of the PFT (FVC, FEV1/FVC, IC, ERV and MVV), RMS (MIP and MEP), CPET (VO2PEAK, VE, RR, OUES and VE/VCO2slope), cardiovascular variables (HR, CO, SAP, SV, total PVR) and metabolic variables (O2Hb, HHb, tHb and VO2PEAK), the two-way mixed ANOVA test of data is parametric and if it is non-parametric, mathematical transformations will be performed in order to normalize the data.
Expected results
Among the scientific contributions from this project, the main one refers to adopting a new evaluation methodology (PThC) and prescription of IMT. It is expected that CPG will obtain better results than 60G and SG, such as obtaining a higher increase in workload (Watts) and peak oxygen uptake (VO2PEAK) in the CPET; higher MIP and iRME; decreased sensation of dyspnea and the sensation of peripheral fatigue, evaluated by the perception index to the physical effort of BORG/CR10 and delay in the activation of the inspiratory muscle metaborreflex both during the CPET and in the iRME.
Thus, it is expected that: using IMT based on this new approach can increase the benefits derived from a traditional methodology (IMT based on 60% of MIP); it can provide subsidies regarding the best understanding of the physiological responses from applying; and this new methodological approach can be used by health professionals as a new tool to evaluate and prescribe IMT, bringing more satisfactory results and greater physiological impact.