The Effects of High Protein Supplementation, Core Muscle Rehabilitation and Neuromuscular Electrostimulation (NMES) Programs on Clinical Outcomes in Patients With Prolonged Mechanical Ventilation (PMV〕
Phase: NA
Conditions: Prolonged Mechanical Ventilation, Protein Deficiency
Sponsor: Chang Gung Memorial Hospital

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1 
 
 
 
 
 
 
CLINICAL STUDY PROTOCOL 
 
〔The effects of high protein supplementation, core muscle 
rehabilitation and neuromuscular electrostimulation (NMES) 
programs on clinical outcomes in patients with prolonged mechanical 
ventilation (PMV)〕 
 
NCT Number: 05932134, Sep 18, 2023 
VERSION NO.1 
 DATES (20221209)  
 
 
INVESTIGATORS 
Chun-Yu Lin 
SITES 
CGMH 
 
 

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Table of Contents 
 
Section 
Page 
1. Synopsis .................................................................................................................................... 1 
2. Introduction and Rationale .............................................................................................................. 
3. Objectives and Endpoints ............................................................................................................ 
3.1. Study Objectives ..................................................................................................................  
3.2. Study endpoints .................................................................................................................  
4. Study Design .............................................................................................................................  
4.1. Overall Design (Flow Chart) ...............................................................................................  
4.2. Number of Patients ................................................................................................................  
4.3. Schedule of Activities (Time-Event scheme) ......................................................................  
5. Study Population .......................................................................................................................  
5.1. Inclusion Criteria ..................................................................................................................  
5.2. Exclusion Criteria ................................................................................................................  
5.3. Withdrawal criteria ..........................................................................................................................  
6. Treatments ................................................................................................................................  
6.1. Treatments Administration ..................................................................................................  
6.2. Concomitant Therapy ......................................................................................................  
7. Efficacy Assessments ...........................................................................................................  
8. Safety Assessments 
9. Adverse Event reporting 
9.1. Definition and reports of Adverse Events ........................................................................  
9.2. Adverse event follow-up ....................................................................................................  
10. Criteria for the termination of the trials 
11. Statistical Considerations 

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11.1. Sample Size Determination ...........................................................................................  
11.2. Planned Statistical methods of analysis .........................................................................  
11.2.1. Efficacy analyses .......................................................................................................  
11.2.2. Safety analyses ..........................................................................................................  
11.2.3. Additional analyses ...................................................................................................  
11.2.4. The level of significance ...........................................................................................  
11.2.5. Analysis Population ..................................................................................................  
12. Direct access to source data/documents 
13. Ethical considerations 
14. Data handling and keeping 
15. Financing and Insurance 
16. References ...............................................................................................................................  

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1. Synopsis 
 
 
Protocol Title：The effects of high protein supplementation, core muscle rehabilitation  and 
neuromuscular electrostimulation (NMES) on clinical outcomes in patients with prolonged 
mechanical ventilation (PMV) 
      
Study Objectives： 
    To evaluate the benefit of high protein supplementation and core muscle rehabilitation and trunk 
NMES intervention for weaning in PMV patients. 
Investigational product(s)： 
 high protein supplementation (1.5 g/kg/day protein) 
 core muscle rehabilitation (sitting for 30 minutes, twice per day, 5 days per week) 
 trunk NMES intervention (Electrodes were placed on back designed to activate  
 latissimus dorsi and abdominal wall designed to activate the transversus abdominis and 
 internal and external oblique muscles.  
Electrical muscle stimulation was performed by using a using a commercial stimulator   
(GEMORE, GM300E, Taipei, Taiwan)    
 with biphasic waves at a simulation frequency of 30 Hz and pulse width of 400s, cycling 2s on   
 and 4s off. Electrical muscle stimulation intensity was gradually increased until a visible muscle  
contraction was observed (median 60 mA [range 50–65 mA].) 
Development Phase：⃞ I   ⃞II    ⃞III     ⃞IV    ⃞其它______  █不適用 
    Study Design： 
1. █ Experimental Group： 
1. High protein supplementation  
2. High protein supplementation + Core muscle rehabilitation 
           3.    High protein supplementation + Core muscle rehabilitation + trunk NMES intervention 
█ Control Group ：  
    Usual care in Respiratory Care Center 
 

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2. Blinding：█ Open  □ Evaluator-blind  □ Single-blind(patient)  □ Double-blind(patient+PI)   
□ Double Dummy   □ Other ______________ 
3.  Randomization:  █ Yes   □ No 
4. □ Parallel design   □ Crossover design   □ Other ____________   █ Not applicable 
5.  Treatment Period：3 weeks  □ Not applicable 
  6.  Study Period: 2 years   
6. Dose adjustment：□ Mandatory     □ Selectively       □ No        █ Not applicable 
7. Study location：█ Single □ Multi-center  □  Global  
Endpoints (Outcome measure)： 
1. Primary endpoint: 
          Weaning rate (weaning success defined as weaning from ventilator for 5 consecutive days) 
2. Secondary endpoints: 
In hospital mortality, Length of mechanical ventilator usage,  
Length of ICU stay and total length of hospitalization,  
Serum albumin level improvement, Creatinine clearance rate 
Inclusion/Exclusion Criteria： 
Patients 
Patients (age ≥ 20 years old) on mechanical ventilator for more than 21 days were recruited from 
the respiratory care center (RCC). of Chang Gung Memorial Hospital in Taiwan.  
 
The inclusion criteria were as follows:  
1. age ≥ 20 years old 
2. using mechanical ventilator for more than 21 days (including patients under tracheostomy or 
endotracheal tube) 
3. stable clinical condition, without using inotropic agent 
(arterial blood gas pH 7.35–7.45, PaO2≥60 mm Hg at FiO2 40%, absence of signs and symptoms 
of uncontrolled infection, and hemodynamic stability) 
4. maximal inspiratory pressure (MIP) < 30mmHg 
5. under enteral nutrition (EN) via NG tube.  

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The exclusion criteria were as follows:  
1. Acute infection and sepsis (fever up to 38.5 degree) 
2. Severe neuromuscular disease, or uncontrolled epilepsy 
3. Bony fracture or DVT history 
4. Wound over the abdomen 
5. Congestive heart failure with EF < 40% or using pacemaker 
6. BMI>35 kg/M2, or severe edema  
7. Patients with hepatic failure, rapid progressed malignancy, or pregnancy were also excluded. 
8. Under parenteral nutrition (PN) 
9. Use pacemaker 
Patient’s identification will be recorded as a study code, which will not be mentioned in the 
publication. 
 
Study Procedures： 
Eligible patients with informed consents will randomly stratify into four groups: (1) Usual care 
(UC), (2) UC + high protein diet (HP), (3) UC + HP + core muscle rehabilitation, (4) UC + HP + 
core muscle rehabilitation + neuromuscular electric stimulation (NMES). Ten patients will be 
assigned to UC group. Thirty patients will be included in another 3 group. An independent 
researcher from Chang Gung Medical Foundation Clinical Trial Center makes random allocation 
cards using computer-generated random numbers. We used sequential sealed envelopes were 
prepared by an independent investigator (Chung-Chi Huang), with one chosen randomly by study 
nurse for each subject. The subjects were then assigned to each group according to the label in the 
envelope. The total daily caloric intake is based on the suggestion of nutritionist. The HP groups 
will maintain unchanged total daily caloric intake and increasing protein content to 1.5g/kg/day. 
The specific formulas were chosen for their different energy and protein requirement (mainly Nutri-
Aid, or NU-PEP HN, according to patients’ digestion, and may add Whey-Aid if necessary (above 
formula are available in CGMH currently)). We will maintain the nutrition strategies for 21 days 
according to patients’ weaning condition. Protein provision was not reduced in case of renal failure. 
Core muscle rehabilitation is sitting on bedside with or without aids, for 30 minutes, twice per day, 
5 days per week, for 3 weeks. NMES was applied for 30 min, twice per day, 5 days per week, for 3 
weeks via surface rectangular electrodes. Electrodes were placed on back designed to activate 
latissimus dorsi and abdominal wall designed to activate the transversus abdominis and internal and 

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external oblique muscles as previously McCaughey et al described 37. Electrical muscle stimulation 
was performed by using a commercial stimulator (GEMORE, GM300E, Taipei, Taiwan) with 
biphasic waves at a simulation frequency of 30 Hz and pulse width of 400s, cycling 2s on and 4s 
off. Electrical muscle stimulation intensity was gradually increased until a visible muscle 
contraction was observed (median 60 mA [range 50–65 mA].  
The age, gender, smoking history, diagnosis, length of mechanical ventilator use, APACHE II will 
be recorded. The pulmonary performance, including tidal volume, maximal inspiratory pressure 
(MIP), cough function, peak inspiratory pressure (PIP), positive end-expiratory pressure (PEEP), 
lung compliance, airway resistance, blood gas analysis, Barthel index, serum albumin, creatinine, 
electrolytes will record before, during (twice per week) and after intervention. We also follow the 
outcome of hospitalization, including the weaning rate, mortality, length of mechanical ventilator 
use, length of ICU stay and total length of hospitalization. We will further evaluate the diaphragm 
movement. Ultrasound was performed at the end of exhalation (without stimulation) to measure the 
thickness of the diaphragm, twice per week of participation, and then weekly until RCC discharge. 
All measurements were taken from muscles on the right-hand side of the participant by the same 
assessor at all assessment sessions. For the diaphragm, the probe was placed parallel to the anterior 
axillary line in the intercostal space between the 9th and 10th rib and moved in the cranial and 
caudal directions until the pleural line was identified. From this point, the probe was moved 
approximately 1 or 2 intercostal spaces lower to identify the costal diaphragm in the zone of 
apposition. In all measurements, the probe was placed perpendicular to the skin. We will analyze 
the diaphragm movement, thickness and the correlation with successful weaning rate.  
 
 

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Concomitant Treatments：  █不適用 
1. Concomitant Therapy： 
      2. Prohibited Therapy： 
Statistical Methods： 
1. Main study Hypothesis： □ Equality  █ Superiority  □ Non-inferiority  
   ⃞ Equivalence   □ Other ________ 
2. Estimated Sample Size：100_ 
3. Efficacy assessment group：  █ Intent-to-treat (ITT)   □ Per-Protocol (PP)   
                                                 □ Other ________ 
  
ITT: We include all eligible patients who meet the criteria had signed informed consents to participate this study. 
 
4. Interim analysis：  □ Yes    █ No 
Statistical methods ：Categorical variables were described using counts (percentages). 
Parametric data were expressed as means ± standard deviation (SD). Among the group 

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comparisons, one-way analysis of variance (ANOVA) followed by Dunnett’s test, where 
appropriate, was used to determine the statistical significance of the difference between means. The 
follow-up parametric data were compared using the paired student’s t-test. Non-parametric data 
were determined by chi-squared tests. Analyses of primary and secondary outcomes are performed 
with the use of time-to-event methods according to the intention-to-treat principle. All analyses 
were two-sided, and p<0.05 was considered statistically significant. Statistical analyses were 
performed using Prism version 5 (GraphPad Software Inc., La Jolla, CA, USA).  
 
5. Handling of Missing Data： 
Omit the cases with missing data and analyze the remaining data.  
 
 
2. Introduction and Rationale 
 
2.1 Investigational product(s) 
       Powered muscle stimulator/ GMORE 
       GM300E  
 
2.2 Animal and preclinical study data 
Not applicable  
 
2.4 Risks / benefits Assessment 
1. In our preliminary study, relative high protein intake (1.2g/kg/day) for less than one month 
had no significant impact on renal function. We will follow serum creatinine level at least twice 
per week for monitoring.  
  
 
2.5 Regulatory 
This study will be conducted in compliance with the protocol approved by the Institutional 
Review Board, and according to Good Clinical Practice standards.  No deviation from the 
protocol will be implemented without the prior review and approval of the IRB except where 

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it may be necessary to eliminate an immediate hazard to a research subject.  In such case, the 
deviation will be reported to the IRB as soon as possible. 
 
3.  Objectives and Endpoints 
3.1 Study Objectives:  
3.1.1 Primary objective: 
          To evaluate the benefit of high protein supplementation and core muscle  
          rehabilitation and trunk NMES intervention in weaning from ventilator 
3.1.2 Secondary objectives: 
          The impact of renal function and digestion status after high protein intake  
          The benefit in nutrition status and diaphragm weakness and the correlation 
          to weaning status 
3.1.3 Other exploratory objectives (if any): 
 
1.2 Study endpoints: 
3.2.1 Primary endpoint: 
         Weaning rate  
         (weaning success defined as weaning from ventilator for 5 consecutive days) 
3.2.2 Secondary endpoints: 
            In hospital mortality, Length of mechanical ventilator usage,  
            Length of ICU stay and total length of hospitalization,  
                      Serum albumin level improvement, Creatinine clearance rate 
            3.2.3 Other exploratory endpoints (if any): 
 
4. Study Design 
 
4.1 Overall Design 
Research principles and methods 
Patients 
Patients (age ≥ 20 years old) on mechanical ventilator for more than 21 days were recruited from 

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the respiratory care center (RCC). of Chang Gung Memorial Hospital in Taiwan.  
 
The inclusion criteria were as follows:  
1. 
age ≥ 20 year old 
2. 
using mechanical ventilator for more than 21 days (including patients under 
tracheostomy or endotracheal tube) 
3. 
stable clinical condition, without using inotropic agent 
4. 
maximal inspiratory pressure (MIP) < 30mmHg 
5. 
under enteral nutrition (EN) via NG tube.  
The exclusion criteria were as follows:  
1. Acute infection and sepsis (fever up to 38.5 degree) 
2. Severe neuromuscular disease, or uncontrolled epilepsy 
3. Bony fracture or DVT history 
4. Wound over the abdomen 
5. Congestive heart failure with EF < 40% or using pacemaker 
6. BMI>35 kg/M2, or severe edema  
7. Patients with hepatic failure, rapid progressed malignancy, or pregnancy were also excluded. 
8. Under parenteral nutrition (PN) 
9. Use pacemaker 
Patient’s identification will be recorded as a study code, which will not be mentioned in the 
publication.  
 
Study Protocol 
Patients will be randomly assigned into four groups: (1) Usual care (UC), (2) UC + high protein 
diet (HP), (3) UC + HP + core muscle rehabilitation, (4) UC + HP + core muscle rehabilitation + 
neuromuscular electric stimulation (NMES). Ten patients will be assigned to UC group. Thirty 
patients will be included in another 3 group. Before subject recruitment, sequential sealed 
envelopes were prepared by an independent investigator (Chung-Chi Huang), with one chosen 
randomly by another investigator for each subject. The subjects were then assigned to each group 
according to the label in the envelope. The total daily caloric intake is based on the suggestion of 
nutritionist. The HP groups will maintain unchanged total daily caloric intake and increasing 
protein content to 1.5g/kg/day. The specific formulas were chosen for their different energy and 

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protein requirement (mainly Nutri-Aid  , or NU-PEP HN  , according to patients’ digestion, and 
may add Whey-Aid   if necessary (above formula are available in CGMH currently)). We will 
maintain the nutrition strategies for 21 days according to patients’ weaning condition. Protein 
provision was not reduced in case of renal failure. Core muscle rehabilitation is sitting on bedside 
with or without aids, for 30 minutes, twice per day, 5 days per week, for 3 weeks. NMES was 
applied for 30 min, twice per day, 5 days per week, for 3 weeks via surface rectangular electrodes. 
Electrodes were placed on back designed to activate latissimus dorsi and abdominal wall designed 
to activate the transversus abdominis and internal and external oblique muscles as previously 
McCaughey et al described 37. Electrical muscle stimulation was performed by using a commercial 
stimulator (GEMORE, GM300E, Taipei, Taiwan) with biphasic waves at a simulation frequency of 
30 Hz and pulse width of 400s, cycling 2s on and 4s off. Electrical muscle stimulation intensity 
was gradually increased until a visible muscle contraction was observed (median 60 mA [range 50–
65 mA].  
The age, gender, smoking history, diagnosis, length of mechanical ventilator use, APACHE II will 
be recorded. The pulmonary performance, including tidal volume, maximal inspiratory pressure 
(MIP), cough function, peak inspiratory pressure (PIP), positive end-expiratory pressure (PEEP), 
lung compliance, airway resistance, blood gas analysis, Barthel index, serum albumin, creatinine, 
electrolytes will record before, during (twice per week) and after intervention. We also follow the 
outcome of hospitalization, including the weaning rate, mortality, length of mechanical ventilator 
use, length of ICU stay and total length of hospitalization. We will further evaluate the diaphragm 
movement. Ultrasound was performed at the end of exhalation (without stimulation) to measure the 
thickness of the diaphragm, twice per week of participation, and then weekly until RCC discharge. 
All measurements were taken from muscles on the right-hand side of the participant by the same 
assessor at all assessment sessions. For the diaphragm, the probe was placed parallel to the anterior 
axillary line in the intercostal space between the 9th and 10th rib and moved in the cranial and 
caudal directions until the pleural line was identified. From this point, the probe was moved 
approximately 1 or 2 intercostal spaces lower to identify the costal diaphragm in the zone of 
apposition. In all measurements, the probe was placed perpendicular to the skin. We will analyze 
the diaphragm movement, thickness and the correlation with successful weaning rate.  
 
Flow Chart  ： 

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14 
 
4.2 
Number of Patients 
  Evaluable Number: 100 
  Enrolled Number: 80 (20% dropped out, or failure to finish study) 
 
4.3 
Schedule of Activities 
 
Time-Event scheme : 

 
15 
 
5. Study Population 
5.1 Inclusion Criteria 
         
The inclusion criteria were as follows:  
1. age ≥ 20 year old 
2. using mechanical ventilator for more than 21 days (including patients under tracheostomy 
or endotracheal tube) 
3. stable clinical condition, without using inotropic agent 
4. maximal inspiratory pressure (MIP) < 30mmHg 
5. under enteral nutrition (EN) via NG tube.  
5.2 Exclusion Criteria 
The exclusion criteria were as follows:  
1. Acute infection and sepsis (fever up to 38.5 degree) 
2. Severe neuromuscular disease, or uncontrolled epilepsy 
3. Bony fracture or DVT history 
4. Wound over the abdomen 
5. Congestive heart failure with EF < 40% or using pacemaker 
6. BMI>35 kg/M2, or severe edema  
7. Patients with hepatic failure, rapid progressed malignancy, or pregnancy were also excluded. 
8. Under parenteral nutrition (PN) 
9. Use pacemaker 
 
5.3 Withdrawal criteria 
1. Return to ICU for unstable vital sign or any necessity of surgery.  
2. Patient refuse further intervention 
3. Physicians judge the patients’ condition are not suitable for further intervention.  
 
 
 
 
10. 
Treatments 

 
16 
 
 
6.1.Treatment Administration 
1. High protein diet: 
The HP groups will maintain unchanged total daily caloric intake and increasing protein content to 
1.5g/kg/day. The specific formulas were chosen for their different energy and protein requirement 
(mainly Nutri-Aid  , or NU-PEP HN  , according to patients’ digestion, and may add Whey-Aid  if 
necessary (above formula are available in CGMH currently)).  
We will maintain the nutrition strategies for 21 days according to patients’ weaning condition. 
Protein provision was not reduced in case of renal failure.  
2. Core muscle rehabilitation: 
Sitting on bedside with or without aids, for 30 minutes, twice per day, 5 days per week, for 3 weeks.  
3. Neuromuscular electrical stimulation (NMES): 
NMES was applied for 30 min, twice per day, 5 days per week, for 3 weeks, via surface rectangular 
electrodes. Electrodes were placed on back designed to activate latissimus dorsi and abdominal 
wall designed to activate the transversus abdominis and internal and external oblique muscles as 
previously McCaughey et al described 37. Electrical muscle stimulation was performed by using a 
commercial stimulator (GEMORE, GM300E, Taipei, Taiwan) with biphasic waves at a simulation 
frequency of 30 Hz and pulse width of 400s, cycling 2s on and 4s off. Electrical muscle stimulation 
intensity was gradually increased until a visible muscle contraction was observed (median 60 mA 
[range 50–65 mA].  
The subjects in the control group received similar electrode placement and intervention duration, 
except that the stimulator power was off. 
 
6.2. Concomitant Therapy 
Not Applicable 
 
       
7. Efficacy Assessments 

 
17 
 
The age, gender, smoking history, diagnosis, length of mechanical ventilator use, APACHE II will 
be recorded initially.  
The pulmonary performance, including tidal volume, maximal inspiratory pressure (MIP), cough 
function, peak inspiratory pressure (PIP), positive end-expiratory pressure (PEEP), lung 
compliance, airway resistance, blood gas analysis, Barthel index, serum albumin, creatinine, 
electrolytes will record before, during (twice per week) and after intervention.  
We also follow the outcome of hospitalization, including the weaning rate, mortality, length of 
mechanical ventilator use, length of ICU stay and total length of hospitalization.  
 
8. Safety Assessments 
We will follow serum creatinine level and creatinine clearance rate weekly. 
During core muscle rehabilitation and NMES, vital signs (heart rate, blood pressure, respiratory 
rate) and parameters in ventilator (tidal volume, FiO2, peak airway pressure) will be monitored.  
 
9. Adverse event reporting 
We will report SAEs to the IRB of Chang Gung Medical Foundation according to the 
Serious Adverse Event Reporting Procedures and Guidelines as posted in the Clinical Trials 
Resource on the website of Chang Gung Medical Foundation IRB. SAE reports to the IRB 
should include the following information when calling the Medical Monitor: 
• Date and time of the SAE 
• Date and time of the SAE report 
• Name of reporter 
• Call back phone number 
• Affiliation/Institution conducting the study 
• Protocol number 
• Title of protocol 
• Description of the SAE, including attribution to drug and expectedness    
 
 
9.1 Definitions and reports of Adverse Events 
All adverse events that occur after the informed consent is signed (including run-in) must be 
recorded on the adverse event CRF (paper and/or electronic) whether or not related to study 

 
18 
 
agent. AE Data Elements including: 
• AE reported date 
• AE Verbatim Term 
• CTCAE Term (v 5.1) 
• Event onset date and event ended date 
• Severity grade 
• Attribution to study agent (relatedness) 
• Whether or not the event was reported as a Serious Adverse Event (SAE) 
• Action taken with the study agent 
• Outcome of the event 
• Comments 
 
Identify the adverse event using the NCI Common Terminology Criteria for Adverse Events 
(CTCAE) version 5.0.  The CTCAE provides descriptive terminology and a grading scale for 
each adverse event listed. 
 
AEs will be assessed according to the CTCAE grade associated with the AE term.  AEs that 
do not have a corresponding CTCAE term will be assessed according to their impact on the 
participant’s ability to perform daily activities as follows: 
 
 
Grade 
Severity 
Description  
1 
Mild 
• 
Barely noticeable, does not influence functioning 
• 
Causing no limitations of usual activities 
2 
Moderate 
• 
Makes participant uncomfortable, influences functioning 
• 
Causing some limitations of usual activities 
3 
Severe 
• 
Severe discomfort, treatment needed 
• 
Severe and undesirable, causing inability to carry out 
usual activities 
4 
Life threatening 
• 
Immediate risk of death 
• 
Life threatening or disabling 
5 
Fatal 
• 
Causes death of the participant 
 
The possibility that the adverse event is related to study drug will be classified as one of the 

 
19 
 
following:  not related, unlikely, possible, probable, definite. 
  
DEFINITION of Serious Adverse Events: ICH Guideline E2A and GCP of Taiwan define 
serious adverse events as those events, occurring at any dose, which meet any of the 
following criteria: 
• Results in death 
• Is life threatening (Note: the term life-threatening refers to an event in which the 
patient was at risk of death at the time of the event; it does not refer to an event 
which hypothetically might have caused death if it were more severe). 
• Requires inpatient hospitalization or prolongation of existing hospitalization 
• Results in persistent or significant disability/incapacity 
• Is a congenital abnormality/birth defect 
• Events that may not meet these criteria, but which the investigator finds very 
unusual and/or potentially serious, will also be reported in the same manner. 
 
9.2 Adverse event follow-up 
All AEs, including lab abnormalities that in the opinion of the investigator are clinically 
significant, will be followed according to good medical practices and documented as such. 
Site staff should send follow-up reports as requested when additional information is available.  
Additional information should be entered on the IRB of Chang Gung Medical Foundation of 
SAE form in the appropriate format.  Follow-up information should be sent to Chang Gung 
Medical Foundation IRB as soon as possible according to IRB’s Serious Adverse Event 
Reporting Procedures and Guidelines.  
 
10. Criteria for the termination of the trial 
          Not applicable. 
 
 
11. Statistical Considerations 
 
11.1 Sample size Determination 
This is an exploratory research, we determine the enrolled number according to the 

 
20 
 
number of patients admitted to RCC annually.  
Ten patients will be assigned to UC group.  
Thirty patients will be included in another 3 group.  
A total 100 patients will be enrolled.  
  
11.2 Planned Statistical methods of analysis 
Categorical variables were expressed as count and percentage. Parametric data were expressed as 
mean ± standard deviation (SD). A Student’s t-test was used to compare parametric data. Fisher’s 
exact test was used to compare non-parametric data. We used a receiver operating characteristic 
(ROC) curve to identify the optimal cut-off-value for the maximum multiplication of sensitivity 
and specificity. Logistic regression was used for multivariate analysis. Kaplan–Meier survival 
analysis was performed to assess weaning and survival outcomes. Statistical significance was set at 
a p-value < 0.05. Statistical analyses were performed using GraphPad Prism version 8 (GraphPad 
Software, La Jolla, CA, USA) and IBM SPSS Statistics 26 (SPSS, Chicago, IL, USA). 
11.2.1 Efficacy analysis 
Kaplan–Meier survival analysis was performed to assess weaning and survival outcomes. 
Mann Whitney test was used for compare non-parametric data (weaning rate, length of ventilator 
use, length of ICU stay and length of hospitalization).  
11.2.2 Safety analysis 
Wilcoxon matched-pairs signed rank test was used to compare parametric data (difference of 
baseline and follow-up serum creatinine and creatinine clearance) 
11.2.3 Additional analysis 
Not applicable 
11.2.4 The level of significance 
A p-value less than 0.05 was considered significant and was denoted by * and p-value 
less than 0.01 was denoted by **. 
 
11.3 Analysis Population 
           All subjects randomized.  
 
11.4 Procedure for accounting for missing, unused and spurious data 

 
21 
 
The incomplete clinical data and sequencing data with poor quality will be excluded in 
the study. 
 
12. Direct access to source data/documents 
Investigators permit IRB to access to the source data of experiment for trial-related 
monitoring, audits and regulatory inspection. 
 
13. Ethical considerations 
            
This study will be conducted according to Taiwan and international standards of Good 
Clinical Practice for all studies.  Applicable government regulations and Chang Gung 
Medical Foundation research policies and procedures will also be followed. 
 
This protocol and any amendments will be submitted to the Chang Gung Medical Foundation 
Institutional Review Board (IRB) for formal approval to conduct the study.  The decision of 
the IRB concerning the conduct of the study will be made in writing to the investigator.   
 
All subjects for this study will be provided a consent form describing this study and 
providing sufficient information for subjects to make an informed decision about their 
participation in this study.  This consent form will be submitted with the protocol for review 
and approval by the IRB.  The formal consent of a subject, using the IRB-approved consent 
form, will be obtained before that subject is submitted to any study procedure.  This consent 
form must be signed by the subject or legally acceptable surrogate, and the investigator-
designated research professional obtaining the consent.  
 
14. Data handling and keeping 
Clinical samples will be collected in Chang Gung Medical Foundation. The sequencing 
data will be stored in computers of laboratory with an electronic encryption. The clinical 
and source data can only be assessed by clinical doctors and investigators of the study. 
 
15.  Financing and Insurance 

 
22 
 
Apply for CMRP support. The funders had no role in the study design, data collection and 
analysis, decision to publish, or manuscript preparation. 
 
16. References 
    
Background 
Ventilator-induced diaphragm dysfunction (VIDD) 
Diaphragmatic function was an important respiratory muscle and play a major role in weaning 
success1. Various insults can weaken the diaphragm, but the most common seen in critically ill 
patients were the use of controlled mechanical ventilation (CMV)1,2. The ventilator assumes the 
entire work of breathing and the diaphragm is for all practical purposes inactive, can rapidly lead to 
atrophy and weakness of the muscle, termed ventilator-induced diaphragmatic dysfunction (VIDD) 
3. Use of mechanical ventilation modes in which the ventilator assists, but does not completely 
supplant, spontaneous diaphragmatic efforts can, to various degrees, attenuate VIDD 1,2.  Reynolds 
et al conducted a study in 18 pigs, use intermittent bilateral phrenic nerve pacing with to prevent 
the decline in diaphragmatic thickness, and to mitigate reductions in diaphragm muscle fiber cross-
sectional area after 60 hours of mechanical ventilation. Maximal pressure generation by the 
diaphragm was not affected, but endurance appeared to be superior (P = 0.055) in the paced group4. 
Is phrenic nerve pacing the sole answer to the problem of VIDD? Further research warranted to 
prove it. Frequent reassessment of the patient’s ability to resume spontaneous breathing and early 
use of partial-support MV are potential strategies to preserve diaphragm protein synthesis during 
prolonged MV 2. Attenuate weakness of diaphragm is an important step in successful weaning.  
Predictors for weaning from prolonged mechanical ventilation 
Several studies have assessed predictors of weaning and extubation outcome in short-term 
mechanically ventilated patients, but there are only few studies on predictors of weaning from 
prolonged mechanical ventilation 5. Female, obesity (BMI>30 kg/m2), hypercapnia, higher tidal 
volume/ideal body weight were independent predictors for weaning failure 5,6. The rapid shallow 
breathing index (RSBI) was introduced by Yang and Tobin in 1991. They found that there was a 
higher probability of weaning success if the RSBI was ≤ 105 and spontaneous breathing trials were 
successful 6-9. In systematic review, Trivedi et al stated that the RSBI has moderate sensitivity and 
poor specificity for predicting extubation success and suggested that it should be a permissive 

 
23 
 
criterion to undergo a spontaneous breathing trial (SBT) for patients who are at intermediate pretest 
probability of passing an SBT 8.  
Since the RSBI represented as respiratory rate/tidal volume (RR/TV), increasing tidal volume is 
quite essential in weaning patients. The factors involving in tidal volume including the VIDD, 
strength of inspiratory muscle and nutritional status.  
Protein supplement strategy in patients with prolonged mechanical ventilation (PMV) 
Critically ill patients undergo severe metabolic stress during which time a great amount of 
energy and protein is utilized in a variety of reactions essential for survival 10. In non-septic 
critically ill patients, early high protein intake (1.2g/kg/d) was associated with lower mortality and 
early energy overfeeding with higher mortality 11. Energy targets are a matter of debate for 
intensive care (ICU) patients. The optimal approach to feeding the critically ill, with increasing 
interest in the concept of intentional underfeeding to reduce metabolic stress while maintaining gut 
integrity 12. In one post hoc–defined outcome, the full energy feeding group was observed to have a 
significantly higher likelihood of being discharged home with or without assistance with activities 
of daily living (68.3% versus 51.3%, P=0.04)13.  Although not reaching statistical significance, full 
energy feeding survivors tended to show improved physical and cognitive function, with a greater 
proportion returning to employment. The full-feeding group also reported a significantly lower 
incidence of admission to physical rehabilitation facilities and trended towards lower new 
residence in healthcare facilities14. A linear reduction in energy target recommendation without 
changing the feed composition led to an unplanned and significant reduction in protein delivery, 
which was associated with a prolonged duration of ventilation and an extended hospital stay 15. 
Thus, during the chronic ICU phase, higher protein/caloric targets should be provided preferably 
combined with exercise 16. Zhang et al conducted a prospective trial discussing the effect of high 
protein supplement (2g/kg/d) on diaphragm atrophy and found that intensive nutrition treatment 
(INT) improved the diaphragm atrophy and muscle mass of critically ill patients receiving 
prolonged MV. There was no evidence that increasing protein to the target amount of 2.0 g/kg/d is 
related to improvement in clinical prognosis for patients receiving prolonged MV 17. The optimal 
protein intake during critical illness is unknown. Conflicting results on nutritional support during 
the first week of ICU stay have been published. Koekkoek et al conducted a retrospective study 
focusing on the timing of protein intake among critically ill patients and found that lowest 6-month 
mortality was associated with patients receiving increasing protein intake from <0.8 g/kg/day on 
day 1-2 to 0.8-1.2 g/kg/day on day 3-5 and >1.2 g/kg/day after day 5 18. Timing of high protein 

 
24 
 
intake may be quite relevant in determining outcomes in ICU patients.  
Rehabilitation in critically ill patients 
Neuromuscular dysfunction in the ICU was common, including demonstrating with flaccid limbs, 
loss of reflexes, and delayed weaning from mechanical ventilation 19. More than 50% of patients 
discharged from the ICU had developed ICU-acquired weakness, which was associated with death 
between ICU discharge and day-90 20. The risk factors for neuromuscular disorders and functional 
impairment were bed rest, corticosteroids, and neuromuscular blockers (NMBs). Early mobilization 
and rehabilitation are quite safe and feasible in patients who are critically ill, with potential benefits 
including improved physical functioning and decreased duration of mechanical ventilation, 
intensive care, and hospital stay 19.  
Attenuate weakness of diaphragm is an important step in weaning. One potential treatment that 
may be useful as an additional form of therapy would be to add muscle-specific forms of exercise. 
Such therapies include various forms of volitional or electrically induced exercise directed at 
various limb and respiratory muscles. In particular, inspiratory muscle training has been shown to 
significantly improve diaphragm function and increase the maximal inspiratory pressure 21. 
Inspiratory muscle training had been proved to be beneficial in patients with COPD and 
bronchiectasis 21,22. Elkins et al systematically reviewed 10 studies involving 394 participants and 
showed that the inspiratory muscle training significantly improved maximal inspiratory pressure, 
the RSBI and weaning success. The significant benefits were also reported for the time spent on 
non-invasive ventilation after weaning and length of stay in hospital. Weaning duration decreased 
in the subgroup of patients with known weaning difficulty 23.  
Furthermore, arranging patient-specific rehabilitation program based upon each patients’ 
functional level, also increased the weaning rate in PMV patients. The multimodal rehabilitation 
program (MRP) is a progressive, patient-specific rehabilitation program, categorized into bed 
dependent, chair dependent and ambulatory groups. MRP improves strength, physical function and 
mobility to usual physical therapy in ICU acquired weakness and was associated with greater 
weaning success and discharge home than UC alone 24. Schreiber et al conducted a retrospective 
analysis on 1,313 consecutive patients admitted to a weaning unit over a 15-y period for prolonged 
mechanical ventilation. Subjects underwent a program of intensive physiotherapy organized in 4 
incremental steps: (1) the ability to maintain a sitting position on the edge of the bed and to 
perform cycling against resistance in bed; (2) the ability to maintain a sitting position in a chair and 
regain standing posture; (3) the ability to start active transfer from bed to chair and to walk with the 

 
25 
 
aid of a rollator and physiotherapists; and (4) the achievement of walking autonomy, with or 
without the aid of a stick and/or a person. Stepwise logistic regression analysis showed that 
achievement of > 2 physiotherapy steps was the main predictor of successful weaning 25. This 
indicated that it is possible that whole body exercise (eg, walking in mechanically ventilated 
patients) may also improve diaphragm strength or endurance.  
Neuromuscular electrostimulation (NMES) 
NMES had played an important role in stroke patients. NMES is also useful for strengthening 
peripheral muscles, augment gains in body weight and quality of life in patients with cystic fibrosis 
with severe pulmonary obstruction 26. NMES could improve exercise capacity and reduce 
perceived sensation of dyspnea during exercise in patients with COPD, but not to be recommended 
as an effective alternative training modality in the rehabilitation of stable COPD patients27-29. 
Home-based NMES as an add-on to PR did not result in further improvements in subjects with 
severe to very severe COPD 30,31. The use of NMES was suggest in patients who were unable to 
engage with conventional pulmonary rehabilitation 29. However, the majority of NMES were 
performed in leg muscles. Zayed et al conducted a systematic review and demonstrated that NES 
combined with usual care was not associated with significant differences in global muscle strength, 
ICU mortality, duration of MV, or ICU length of stay in comparison with usual care alone in 
critically ill patients 32. But Leite et al showed electrical stimulation of quadriceps had best 
outcomes for peripheral muscle strength compared with controls or electrical stimulation of 
diaphragm among mechanically ventilated critically ill subjects and promoted functional 
independence and decreased length of hospitalization 33. The effects of limbs NMES remained 
controversial.  
Recently, WB-EMS is proved to be a safe and attractive method for increasing muscle mass and 
functional capacity in this cohort of women 70+ with sarcopenic obesity 34. Core muscle 
strengthening combined with trunk NMES improved on trunk balance in post-stroke patients 35 and 
the effects were similar in abdominal and back muscles 36. Since the diaphragm played an 
important role in respiration and weaning. McCaughey et al preformed a prospective study in 20 
critically ill mechanically ventilated participants. Electrodes were placed posteriorlaterally over the 
abdominal wall designed to activate the transversus abdominis and internal and external oblique 
muscles. The active group received abdominal FES at an intensity that caused a strong visible 
muscle contraction (median 60 mA [range 50–65 mA]), with a frequency of 30Hz and a pulse 
width of 350 μs. The stimulation current in the control group was set at 10 mA (possible sensation 

 
26 
 
but no muscle contraction), with a frequency of 10Hz and a pulsewidth of 350 μs. There appeared 
to be no between-group thickness changes of the rectus abdominis, diaphragm or combined lateral 
abdominal muscles. However, ICU length of stay and ventilation duration appeared to be shorter in 
the intervention compared to the control group 37. Cho et al conducted a prospective trial discussing 
the effects of NMES in different frequencies and demonstrated that 50 Hz produced best muscle 
thickness increases 38. Sewa et al revealed that functional electrical stimulation can be safely 
delivered to human abdominal muscles without causing vital sign abnormalities. It was also found 
that the appropriate intensity level of electrical stimulation for achieving effects on respiratory flow 
while also minimizing pain is 60–80 mA39. The effects of NMES on abdominal muscles and 
diaphragm were not clear.  
Overall, the effects of protein nutrition, exercise, NMES appear to be beneficial in critically ill 
patients. However, studies into the differential effects of protein nutrition and/or exercise combined 
with trunk NMES, and optimization of their combined use, have not been performed yet 40. 
 
Aims 
The aim of this study is to investigate the outcomes of combined high protein supplementation 
and NMES on truck muscles in patients with prolonged mechanical ventilation. 
Importance 
Analyzing the role and timing of high protein supplementation and trunk NMES intervention will 
provide a more precise treatment strategy in future for weaning in PMV patients. 
 
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