LEan Body Mass Development in CriTically Ill Patients, Obtained by bioelectRical impEdance Analysis: a Combined Retrospective and Prospective Observational Cohort Study
Conditions: Critical Illness
Sponsor: Gelderse Vallei Hospital

 
 
 
 
 
 
 
Study Protocol 
 
 
Protein Dosing Strategies in Critically Ill Patients: Comparing 
Actual Body Weight and Fat-Free Mass Approaches in an 
Observational Cohort Study 
 
 
Protocol date: 1 March 2023, revised 20 October 2025 
 
File number: 2403-017 
 
 
 
 
 
 
 
 
Lead clinical investigator:  
Michelle Carmen Paulus, MD, Ph.D. candidate, Department of Intensive Care, Gelderse Vallei 
Hospital, Ede, The Netherlands, 
Email: mpaulus@zgv.nl 
 
Supervisor and Principal investigator 
Prof. Dr. Arthur R.H. van Zanten, MD, PhD 
Internist-Intensivist 
Chair Department of Intensive Care & Research 
Intensive Care Staf Route 61 A3 (+31-318-434115) 
Gelderse Vallei Hospital 
Willy Brandtlaan 10, 6716 RP Ede, The Netherlands 
Tel. +31 (318) 43 4343 
www.geldersevallei.nl 
E-mail: zantena@zgv.nl 
Wageningen University & Research 
Division of Human Nutrition and Health 
HELIX (Building 124), Stippeneng 4 | 6708 WE Wageningen, The Netherlands 
Email: arthur.vanzanten@wur.nl 
 
 
 
 
 

Protocol synopsis 
 
Study Title 
Protein Dosing Strategies in Critically Ill Patients: Comparing Actual Body 
Weight and Fat-Free Mass Approaches in an Observational Cohort Study 
 
Study 
Objectives 
The study is designed to investigate the following research questions: 
Primary: 
The primary outcome measure of this study is 90- day mortality, comparing 
patients who received protein based on total body weight with those who received 
protein based on fat-free mass. Mortality will be assessed as both short-term 
mortality (ICU and 30-day mortality) and long-term mortality (90-day mortality). 
 
Secondary: 
Secondary outcome measures include several clinical and nutritional endpoints, 
all comparing the cohort receiving protein based on actual body weight with the 
cohort receiving protein based on fat-free mass: 
• 
ICU and hospital length of stay, defined as the total duration of ICU and 
hospital admission in days, measured from ICU admission until discharge. 
• 
Duration of invasive mechanical ventilation (IMV), defined as the 
number of days a patient receives IMV from initiation until 
discontinuation. 
• 
Urea–creatinine ratio (UCR) during ICU stay, comparing differences 
in UCR between both groups throughout the ICU stay. 
• 
Changes in BIA parameters over time, including changes in fat-free 
mass and other body composition measures, based on repeated 
bioelectrical impedance analysis (BIA) measurements during ICU stay. 
• 
Protein and energy intake during ICU stay, comparing daily and 
cumulative (first 14 days) protein and energy intake between the two 
groups. 
• 
Hazard ratio for mortality, evaluating the relationship between protein 
intake (based on TBW and FFM) and mortality. This includes actual 
protein intake per kilogram TBW and FFM, with analysis of days with 
full intake and patients with an ICU stay >7 days. Covariates will be 
included to account for potential confounders. The analysis will also be 
broken down by sex.  
• 
Differences in indirect calorimetry between the two groups, assessing 
energy expenditure and comparing results from indirect calorimetry 
measurements during ICU stay. 
 
Study Design 
This study is a single-centre, combined retrospective and prospective cohort study 
conducted at Hospital Gelderse Vallei. Since April 2020, measurement of body 
composition has been implemented as part of standard care for all patients 
admitted to the intensive care unit (ICU). Each patient admitted to the ICU 
undergoes bioelectrical impedance analysis (BIA) upon admission and 
approximately every third day thereafter (±1 day), using the InBody S10® device 
(InBody Co., Ltd., Seoul, Korea). 
From July 7th, 2023, protein requirements for ICU patients have been calculated 
based on fat-free mass rather than total body weight, as part of the standard of 
care. This study evaluates differences in clinical outcomes resulting from this 
change in practice. 

All patients are screened for inclusion and exclusion criteria upon ICU admission. 
BIA measurements are extracted directly from the device. Data on protein intake 
and clinical outcome measures are retrieved and anonymized from the electronic 
patient record systems MetaVision® (iMDsoft, Tel Aviv, Israel), Neozis® (MI 
Consultancy, Katwijk, The Netherlands), and Nexus® (NEXUS AG, 
Donaueschingen, Germany). 
 
Patients 
620 critically ill patients admitted to the ICU (310 with protein dosing based on 
actual body weight and 310 with protein dosing based on fat free mass) 
 
Inclusion 
Criteria 
Patients meeting all of the following criteria are eligible for the study:  
1. Age ≥ 18 years 
2. A BIA measurement within 48 hours after ICU admission 
3. Two BIA measurements during ICU stay 
 
Exclusion 
Criteria 
Any patient meeting one or more of the following criteria is not eligible for the 
study: 
1. Transfer from another ICU resulting in the first BIA measurement >48 
hours after admission to Gelderse Vallei Hospital 
2. A previous ICU admission within 2 weeks before the current admission 
3. Contra-indications for BIA measurement:  
i. 
Patients with electrical implants such as a pacemaker or ICD 
ii. 
Patients who cannot maintain posture for the (5 min) duration of 
the measurement 
iii. 
Patients with wounds or skin damage in the designated electrode 
sight 
iv. 
Pregnancy 
Study 
Parameters 
The objective parameters to be studied will be: 
• 
All-cause 90-day mortality  
• 
All-cause 30-day mortality 
• 
All-cause in-hospital and ICU mortality 
• 
Hospital length of stay  
• 
ICU length of stay 
• 
Post-ICU hospital days 
• 
Time-to-discharge alive 
• 
Length of mechanical ventilation  
• 
Body composition measured through body impedance analysis upon 
admittance and every third day (+/- 1 day) after that   
• 
Urea-to-creatinine ratio during ICU stay 
• 
Daily protein intake during ICU stay 
• 
Daily energy intake during ICU stay 
• 
Protein target during ICU stay 
• 
Energy target during ICU stay 
Other 
Parameters 
Demographical characteristics  
• 
Age [yrs], race, sex [m/f], weight [kg], height [cm], and weight/height 
(BMI in kg/m2) 
• 
Pre-existing comorbidities 
• 
Type of admission (medical, surgical/emergency or elective), covid-19 at 
admission.  
• 
Baseline laboratory results (CRP, albumin, creatinine, blood urea nitrogen 
(BUN), urea-to-creatinine-ratio 
Markers of disease severity and treatment 

• 
ICU admission diagnosis 
• 
APACHE-II and IV scores 
• 
NUTRIC-score 
• 
Barthel Score 
• 
SOFA-score 
Study Group 
All patients admitted to the ICU will be included according to the in- and 
exclusion criteria. A total of 620 eligible patients will be included: 310 treated 
during the ABW dosing period and 310 during the FFM dosing period 
Study Period 
The inclusion period began in April 2020, when BIA (Bioelectrical Impedance 
Analysis) became part of standard care. A total of 620 patients will be included in 
the study. One group of patients was fed based on ABW (Actual Body Weight) 
until July 2023, and a second group was fed based on FFM (Fat-Free Mass) 
thereafter. Patients for whom obtaining informed consent was not feasible were 
included retrospectively, while prospective inclusion continued afterward. The 
study will conclude once a total of 620 patients have been included, followed by a 
90-day follow-up period for the final patient 
Study 
Description 
This study aims to evaluate the effect of two different methods of calculating 
protein requirements on clinical outcomes and body composition in critically ill 
patients admitted to the Intensive Care Unit (ICU) at Hospital Gelderse Vallei. 
Since April 2020, bioelectrical impedance analysis (BIA) has been implemented 
as standard practice to assess body composition in all ICU patients. In July 2023, 
the ICU transitioned from protein dosing based on actual body weight to dosing 
based on fat-free mass. The study compares these two cohorts to determine 
whether protein dosing based on fat-free mass results in improved clinical 
outcomes, such as lower mortality, shorter ICU and hospital length of stay, 
reduced duration of mechanical ventilation, preservation of muscle mass, and 
lower urea–creatinine ratios. All patients admitted to the ICU will be screened for 
inclusion and exclusion criteria. Those meeting all inclusion and no exclusion 
criteria will be enrolled in the study. Data on body composition, protein intake, 
and clinical outcomes will be collected and analysed. 
 
Statistical 
Analysis 
All statistical analyses will be conducted using R 4.4.1 and Rstudio. Continuous 
variables will be reported as means or medians, when appropriate, including SD, 
ranges, and 95%CI. Discrete variables will be displayed as proportions. P-values 
<0.05 will be considered statistically significant. Comparisons will be made using 
appropriate statistical tests.  
 
 
 
 
 
 
 
 
 
 
 
 

Table of Contents  
1 Introduction 
1.1 Background 
1.2 Description of the study 
1.3 Rationale for study design 
2 Study Objectives 
2.1 Primary study objective 
 
2.2 Secondary study objective 
3 Study Design 
3.1 Study description 
3.2 Schematic diagram of trial design 
3.3 Schedule of assessments 
3.4 Duration of the study per patient 
4 Patients 
 
4.1 Study population 
 
4.2 Inclusion criteria 
 
4.3 Exclusion criteria 
 
4.4 Patient recruitment 
 
4.5 Instructions and restrictions during the study 
 
4.6 Patient discontinuation during the study 
 
 
4.6.1 Criteria for patient discontinuation 
 
 
4.6.2 Procedures in case of patient discontinuation 
5. Study parameters 
 
5.1 Primary study parameter 
 
5.2 Secondary study parameters 
 
5.3 Safety and tolerance parameters 
 
5.4 Other study parameters and covariates 
6. Study procedures and assessments 
 
6.1 Study procedures 
 
 
6.1.1. Screening 
 
 
6.1.2. Measurements 
 
6.2 Assessments 
 
 
6.2.1. Demographics 
 
 
6.2.2. Anthropometry 
 
 
6.2.3. Medical history, pre-existing conditions, and concomitant medication 
 
 
6.2.4. Diagnosis 
 
 
6.2.5. Clinical measurements 
 
 
6.2.6. Safety measurements 
 
 
7. Statistics 
8. Ethical considerations 
 
8.1 Basic principles and regulations 
 
8.2 Ethics Committee 
 
8.3 Informed Consent 
 
8.4 Confidentiality of study data 
9. Administrative aspects 
 
9.1 Source data 
 
9.2 Data handling 
 
9.3 Documentation and storage 
10. References 
 
 
 
 
 

Abbreviations and Definitions 
 
Term 
Explanation 
BIA 
Bioelectrical Impedance Analysis 
BMI 
Body Mass Index 
EC 
Ethics Committee 
FFM 
Fat-Free Mass 
IBW 
Ideal Body Weight 
ICU 
LBM 
TBW 
Intensive Care Unit 
Lean Body Mass 
Total Body Weight 
ZGV 
Ziekenhuis Gelderse Vallei 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

1 Introduction 
1.1 Background 
Proteins are essential macronutrients for critically ill patients. Deficiency of proteins can result in a 
higher rate of hospital-acquired infections, impaired wound healing, and loss of skeletal muscle mass 
(1,2). On the other hand, adequate protein intake during ICU admission is associated with a higher 
degree of recovery to independent walking (3), which can most likely be translated into preserved 
muscle strength at ICU discharge.  
Critically ill patients are known to be prone to sarcopenia, which can be defined as generalized and 
progressive loss of muscle mass and strength. (4) Sarcopenia can be developed due to different factors 
such as inactivity, inflammation, and nutritional status.(5) The prevalence of sarcopenia is estimated to 
be approximately 30-70% in patients admitted to the intensive care unit (ICU) and is associated with 
adverse clinical outcomes and even mortality.(5,6). 
 There are studies performed that have shown beneficial effects on the loss of muscle mass and muscle 
protein synthesis induced by the administration of higher dosages of protein.(7) An element of 
establishing the appropriate dosages of protein is providing insight into the influence of protein 
provision on this process. This can be carried out by repeatedly measuring the lean body mass (LBM) 
from ICU admission until discharge.  
In recent years, the importance of including body composition in assessing a patient's nutritional status 
has become increasingly apparent (8,9). Our hypothesis, supported by studies like Velzeboer et al. 
2017 (10), is that protein requirements are primarily determined by the fat-free mass (FFM) of the 
body. However, in actual practice protein goals are often calculated using the actual body weight or 
estimated ideal body weights with predictive formulas. Predictive formulas cannot take real-time 
changes in muscle mass or sarcopenic obesity into account, and can differ greatly from the actual lean 
body mass.  This is especially a problem when people are underweight or overweight. (10)  
 
There are several methods to determine the LBM, including dual-energy X-ray absorptiometry 
(DXA/DEXA), computerized tomography (CT), and magnetic resonance imaging (MRI)[11]. 
Disadvantages of these techniques include the need for specialized trained personnel, high costs, 
stationary equipment not available in the ICU itself, the time-consuming process, and the possible 
involvement of radiation exposure (12, 13). Another method to determine the LBM is using 
bioelectrical impedance analysis (BIA). BIA is a technique in which a weak current is passed through 
the body, and the resistance across this current is calculated. The advantage of BIA over other methods 
is that it is non-invasive, less expensive, and fast(14,1). Moreover, it can be used in the ICU due to its 
portability(16). These BIA measurements could be an alternative to the before-mentioned techniques 
to estimate the LBM. 
 
1.2 Description of the study 
This study is a single-centre combined retrospective and prospective cohort study, performed at 
hospital Gelderse Vallei. Starting from April 2020, the measurement of body composition of all 
patients admitted to the intensive care unit in our hospital was implemented as a standard of care. All 
patients admitted to the intensive care unit receive a bioelectric impedance analysis (BIA) upon 
admittance and every third day (-/+ 1 day) after that, using the Inbody S10 ® (InBody Co., Ltd., Seoul, 
Korea).  
Every patient will be screened in- and exclusion criteria upon admittance. The BIA measurements will 
be extracted from the device. The patients' protein intake and various clinical outcome measures will 
be extracted from the electronic patient record MetaVision ® (iMDsoft, Tel Aviv, Israel), Neozis and 
Nexus and anonymized. Data will be extracted, and the database will be locked after the protocol 
publication. 
 
 
 

Rationale for study design 
This study hypothesizes that calculating protein requirements based on fat-free mass rather than actual 
body weight will result in improved clinical and nutritional outcomes in critically ill patients. Current 
clinical practice often relies on protein dosing per kilogram of actual body weight, which does not 
account for variations in body composition such as sarcopenia or obesity. Using fat-free mass provides 
a more accurate reflection of the metabolically active tissue and may therefore allow for more precise 
and individualized protein dosing. Prospective data on changes in lean body mass (LBM) among ICU 
patients—measured using bioelectrical impedance analysis (BIA) while correcting for extracellular 
fluid—are currently lacking. This study addresses that gap by systematically monitoring body 
composition throughout ICU admission. The findings may offer valuable insight into muscle mass 
deterioration during critical illness and support the development of more accurate protein requirement 
guidelines.  
 
2 Study objectives 
Primary: 
The primary outcome measure of this study is 90-day mortality, comparing patients who received 
protein based on total body weight with those who received protein based on fat-free mass. Mortality 
will be assessed as both short-term mortality (ICU and 30-day mortality) and long-term mortality (90-
day mortality)Secondary: 
Secondary outcome measures include several clinical and nutritional endpoints, all comparing the 
cohort receiving protein based on actual body weight with the cohort receiving protein based on fat-
free mass: 
• 
ICU and hospital length of stay, defined as the total duration of ICU and hospital admission 
in days, measured from ICU admission until discharge. 
• 
Duration of invasive mechanical ventilation (IMV), defined as the number of days a patient 
receives IMV from initiation until discontinuation. 
• 
Urea–creatinine ratio (UCR) during ICU stay, comparing differences in UCR between both 
groups throughout the ICU stay. 
• 
Changes in BIA parameters over time, including changes in fat-free mass and other body 
composition measures, based on repeated bioelectrical impedance analysis (BIA) 
measurements during ICU stay. 
• 
Protein and energy intake during ICU stay, comparing daily and cumulative (first 14 days) 
protein and energy intake between the two groups. 
• 
Hazard ratio for mortality, evaluating the relationship between protein intake (based on 
TBW and FFM) and mortality. This includes actual protein intake per kilogram TBW and 
FFM, with analysis of days with full intake and patients with an ICU stay >7 days. Covariates 
will be included to account for potential confounders. The analysis will also be broken down 
by sex.  
• 
Differences in indirect calorimetry between the two groups, assessing energy expenditure 
and comparing results from indirect calorimetry measurements during ICU stay. 
 
3 Study design 
This is a combined retrospective and prospective observational cohort study. 
 
3.1 Study description 
Upon admittance, all patients will be screened according to the in- and exclusion criteria on ICU 
admission. Patients that meet all the inclusion criteria and none of the exclusion criteria will be 
included in this prospective observational cohort study. Patients will be enrolled through an opt-out 
procedure with a leaflet in the admission folder.  
 
Height and weight will be measured and recorded upon admittance by the researchers. Measurements 
will be performed by ICU nurses following standard of care within 24 hours after admittance to the 
ICU and repeated every 3 days. The BIA measurements will be extracted from the device. Various 
baseline variables, daily protein and energy intake (both enteral and oral), and various clinical 

outcome measures will be extracted from the electronic patient record MetaVision ® (iMDsoft, Tel 
Aviv, Israel) and Nexus and anonymized.  
 
3.2 Schematic diagram of study design 
 
 
 
4 Patients  
4.1 Study population 
Critically ill patients admitted to the ICU 
4.2 Inclusion criteria  
Patients meeting all of the following criteria are eligible for the study:  
1. Age ≥ 18 years 
2. A BIA measurement within 48 hours after ICU admission 
3. Two BIA measurements during ICU stay 
 
4.3 Exclusion criteria 
Any patient meeting one or more of the following criteria is not eligible for the study: 
1. Transfer from another ICU resulting in the first BIA measurement >48 hours after admission 
to Gelderse Vallei Hospital 
Inclusion of patients according to 
in-and exclusion criteria 
Obtain coded and anomysed data
Publish Statistical Analysis Plan 
Data analysis
Write final report

2. A previous ICU admission within 2 weeks before the current admission 
3. Contra-indications for BIA measurement:  
v. 
Patients with electrical implants such as a pacemaker or ICD 
vi. 
Patients who cannot maintain posture for the (5 min) duration of the measurement 
vii. 
Patients with wounds or skin damage in the designated electrode site 
viii. 
Pregnancy 
 
5 Study parameters 
5.1 Objective study parameters  
The objective parameters to be studied will be: 
 
• 
All-cause 90-day mortality  
• 
All-cause 30-day mortality 
• 
All-cause in-hospital and ICU mortality 
• 
Hospital length of stay  
• 
ICU length of stay 
• 
Post-ICU hospital stay 
• 
Time-to-discharge alive 
• 
Length of mechanical ventilation (invasive and non-invasive) 
• 
Body composition measured through body impedance analysis upon admittance and every 
third day (+/- 1 day) after that   
 
• 
Urea-to-creatinine ratio during ICU stay 
• 
Daily protein intake during ICU stay (absolute and per kg IBW and FFM). IBW: TBW is used 
if BMI < 27; if BMI > 27, weight is calculated for a BMI of 27 
• 
Daily energy intake during ICU stay (absolute and per kg IBW and FFM). IBW: TBW is used 
if BMI < 27; if BMI > 27, weight is calculated for a BMI of 27 
• 
Protein target during ICU stay 
• 
Energy target during ICU stay 
 
5.2 Overview of study parameters and covariates  
Demographical characteristics  
• 
Age [yrs], race, sex [m/f], weight [kg], height [cm], and weight/height (BMI in kg/m2) 
• 
Pre-existing comorbidities 
• 
Type of admission (medical or surgical and emergency or elective).  
• 
Baseline laboratory results (CRP, albumin, creatinine, blood urea nitrogen (BUN), urea-to-
creatinine ratio) 
 
Markers of disease severity and treatment 
• 
ICU admission diagnosis 
• 
APACHE-II and IV scores 

• 
Barthel score 
• 
NUTRIC-score 
• 
SOFA-score 
 
6 Study procedures and assessments 
6.1 Study procedures  
6.1.1 Screening 
The inclusion period started on April 2020. Patients for whom obtaining informed consent was not 
feasible (before BCWO approval) were included retrospectively, while prospective inclusion 
continued thereafter with opt-out procedure. The study will conclude once a total of 620 patients have 
been included, followed by a 90-day follow-up period for the final patient. 
 
6.1.2. Measurements 
BIA measurements will be performed by the ICU nurses within 24 hours of admittance and repeated 
every third day (+/1 day) after that during ICU admission.  
 
6.2 Assessments 
6.2.1 Demographics 
Demographics including date of birth (age) and gender are recorded.  
 
6.2.2 Anthropometry 
Actual body weight and height will be measured by the researchers. Bodyweight and height are used 
to calculate the Body Mass Index (BMI, kg/m2). 
 
6.2.3 Medical history, pre-existing conditions, and concomitant medication 
Relevant medical history and pre-existing conditions are recorded at baseline. 
 
6.2.4 Diagnosis 
Diagnosis for admission will be recorded at screening.  
 
6.2.5 Clinical measurements 
BIA measurements will be performed by the ICU nurses within 24 hours and repeated every third day 
(+/1 day) after that during ICU admission (standard care).  
 
7. Statistical analysis plan 
7.1 General considerations 
 
All statistical analyses will be performed using R version 4.4.1 (or updated version). Categorical data 
will be presented as counts alongside percentages. Continuous data will be presented as either mean 
with standard deviation or median with interquartile range [IQR] in case of non-normal distribution. 
Baseline and outcome differences will be compared using the Chi-Square or Fisher exact test for 
categorical variables, independent sample t-test for normally distributed continuous data, and Mann-
Whitney U test for continuous data with non-normal distributions. Linearity over time will be assessed 
visually and by comparing model fit between linear and spline-based mixed models. Non-linear 
models will be chosen if they significantly improve model fit. 
 
7.2 Primary analysis 
The primary endpoint is 90-day mortality. Mortality will be compared between patients receiving 
protein prescribed according to total body weight (TBW cohort) and those receiving protein based on 
fat-free mass (FFM cohort). Unadjusted cumulative survival will be visualized using Kaplan–Meier 
curves, and group differences will be evaluated using the log-rank test. To estimate independent 

associations between protein dosing method and 90-day mortality, multivariable Cox proportional 
hazards regression models will be fitted. Covariates will be eligible for inclusion only if <10% of their 
values are missing. Candidate variables will first be screened using univariable Cox regression; 
variables associated with mortality at p < 0.20 will be considered for the multivariable model. Baseline 
characteristics that differ between groups or are clinically important will also be evaluated and 
included if they improve model fit. 
Potential covariates include age, sex, APACHE IV score, admission category (medical/surgical), 
COVID-19 status, UCR at admission, and dry fat-free mass (dryFFM). Collinearity will be assessed 
using Variance Inflation Factors (VIF > 5 indicating concern). Proportional hazards assumptions will 
be tested using Schoenfeld residuals; if violated, models with time-varying effects or stratified Cox 
models will be considered. Covariate numbers will follow the one-in-ten rule to limit overfitting. 
Results will be reported as hazard ratios (HRs) with 95% confidence intervals (CIs), using two-sided p 
< 0.05 as significance threshold. 
If substantial baseline imbalance is identified, a sensitivity analysis using propensity scores will be 
performed with relevant covariates (age, sex, APACHE score, BMI, admission category). 
7.3 Secondary Endpoint Analyses 
7.3.1 Continuous single-measurement outcomes 
ICU length of stay, hospital length of stay, and duration of invasive mechanical ventilation will be 
compared using linear regression. Models will be adjusted for baseline covariates that differ between 
groups or are clinically relevant (Age, APACHE IV , Covid, dryFFM). 
7.3.2 Repeated-measure outcomes (UCR, BIA parameters) 
Longitudinal outcomes, including the urea–creatinine ratio (UCR) and dryFFM, will first be visualized 
graphically to assess temporal patterns. If appropriate, linear mixed-effects models (LMMs) with a 
random intercept for participants will be used. Time will be included as a categorical or spline-based 
variable depending on observed non-linearity. Interaction terms between group and time will be tested 
to evaluate differential evolution across cohorts. 
7.4 Missing Data 
The primary outcome (mortality) is expected to be complete; no imputation will be performed. 
Secondary outcomes with missingness (e.g., laboratory values, BIA measures, nutritional intake) will 
be assumed missing at random. If missing data are substantial, multiple imputation sensitivity analyses 
will be performed. 
7.5 Additional Analytic Procedures (Study-Specific Specifications) 
7.5.1 UCR change-from-baseline (days 1–30) 
Daily UCR will be analyzed from day 1 to day 30. 
• 
UCR will be included only up to the initiation of dialysis (CVVH). 
• 
Patients with active gastrointestinal bleeding will be excluded from UCR analyses. 
• 
UCR change will be expressed as percentage change relative to baseline. 
• 
Longitudinal modeling will use linear mixed-effects models; non-linearity will be 
accommodated via spline functions. 
• 
Sex interactions will be evaluated, and stratified figures will be produced for men and women. 

• 
Analyses will be repeated for all patients and for the non-COVID subgroup. 
7.5.2 Body weight definitions 
• 
For BMI < 27 kg/m², total body weight (TBW) will be used as recorded. 
• 
For BMI ≥ 27 kg/m², TBW will be adjusted downward to the weight corresponding to BMI 27 
to create an ideal body weight (IBW) equivalent for intake calculations. 
7.5.3 Computation of dry fat-free mass (dryFFM) 
Fluid overload will be derived as: 
FO = ECW − (0.38 × ICW / 0.62) 
If FO > 0, this value will be subtracted from FFM to calculate dryFFM. 
7.5.4 Nutritional intake calculations 
Daily nutritional intake will be calculated as the sum of oral and enteral intake. 
• 
Intake will be expressed separately for protein and energy. 
• 
Non-nutritional calories (e.g., from citrate and propofol) will be included in total energy 
intake. 
• 
Days without recorded nutritional data will be treated as missing. 
• 
Discharge day will be excluded; admission day will be included, with intake target scaled to 
the actual hours of observation. 
• 
A standardized buildup protocol will be applied, increasing intake by 25% per 24 hours until 
full target is reached on day 4. 
• 
In case of refeeding syndrome, defined as a decrease in phosphate from >0.70 mmol/L at 
admission to <0.65 mmol/L within 72 hours without another cause, targets will be reset to 
25% for 48 hours, after which increments of 25% per day resume. 
7.5.5 Protein dosing and intake metrics 
No a priori difference in baseline characteristics is expected other than the dosing strategy: 
• 
TBW cohort: prescribed at 1.5 g/kg TBW aiming for 1.3 g/kg (ESPEN) for patients with 
normal BMI, if BMI >27 IBW is used. 
• 
FFM cohort: prescribed at 1.85 g/kg dryFFM. 
Daily intake and cumulative intake will be expressed per kg IBW and per kg FFM. 
Separate figures will depict protein and energy intake normalized by TBW (accounting for IBW) and 
by FFM, stratified by sex. 
7.6 Baseline and Outcome Tables 
7.6.1 Baseline characteristics 
Baseline tables will include all prespecified binary and continuous variables. 
• 
Continuous variables will be summarized using mean (SD) or median [IQR] depending on 
distribution. 

• 
Statistical testing: t-tests for normally distributed variables, Wilcoxon tests for non-normal 
variables, and χ² tests for binary variables. 
Baseline tables will be generated for all patients and non-covid patients 
• 
Outliers will not be removed unless reflecting data entry errors. 
7.6.2 Outcome tables 
Binary and continuous outcomes will be summarized similarly and compared between groups. 
Outcome tables will be produced for all patients and non-covid patients. Outliers will not be removed 
unless reflecting data entry errors. 
7.7 Primary outcome subgroup analyses 
A separate baseline table will be produced comparing 90-day survivors and non-survivors across all 
baseline variables. Kaplan–Meier curves will be generated to compare unadjusted survival between 
cohorts. A final Cox model including age, APACHE IV, UCR, cohort, COVID status, and dryFFM 
will be fitted, with assessment of collinearity and proportional hazards assumptions. 
Adjusted survival curves will be generated from model-based predictions. 
7.8 Sensitivity analysis: Actual protein intake and 90-day mortality 
Among patients with ICU length of stay ≥ 7 days, an analysis will focus on days where ≥100% of the 
prescribed target was achieved (excluding buildup/refeeding days). Protein intake will be expressed 
per kg TBW (accounting for IBW) and per kg FFM. 
Restricted cubic spline Cox models will be used to evaluate dose-response shapes for protein intake, 
adjusted for age, APACHE IV, COVID status, and UCR. Sex-stratified figures will be created. 
7.9 Body composition analyses 
7.9.1 Temporal evolution of BIA parameters 
Figures will be created for: 
• 
All patients 
• 
ICU survivors only 
DryFFM percentage change relative to baseline will be analyzed. 
• 
If changes appear linear, linear mixed-effects models will be used. 
• 
If non-linear, spline-based mixed-effects models will be fitted. 
• 
Interaction with cohort will be evaluated. 
7.9.2 Relationship between TBW and FFM at baseline 
Scatterplots with LOESS smoothing will show the association between BIA-measured TBW and 
dryFFM, with separate curves for men and women and an overall dashed curve. The potential for 
deriving sex-specific conversion factors to estimate FFM from TBW will be explored. 
7.10 UCR longitudinal analyses (detailed) 
UCR percentage change from baseline will be analyzed from day 1–30. Analyses will be performed 
for all patients and non-COVID patients.  

Mixed-effects spline models will include cohort × time interactions, and additional sex interactions 
where appropriate. Both percentage change and absolute UCR values may be presented graphically. 
 
7.11 Indirect calorimetry (REE) analyses 
Resting Energy Expenditure (REE) measured by indirect calorimetry will be analyzed longitudinally 
using mixed-effects spline models, adjusting for age, sex, and dryFFM. Predicted curves over time 
will be plotted for each cohort. 
 
8 Ethical considerations 
8.1 Basic principles and regulations 
The investigator will ensure that this study is conducted in full conformance with the principles of the 
‘World Medical Association Declaration of Helsinki’ (52nd WMA General Assembly, Edinburgh, 
Scotland, October 2000, including the Notes of Clarification as added in 2002, Washington, and 2004, 
Tokyo).  
 
8.2 Ethics Committee 
This protocol was deemed not subject to the Dutch Medical Research Involving Human Subjects Act 
(niet-WMO). Therefore, approval from a central ethics committee was not required. The protocol was 
reviewed and approved by the Hospital Gelderse Vallei internal research review committee (BCWO). 
 
8.3 Informed Consent 
To minimize the risk of consent bias and reduce the burden on critically ill patients and their families, 
an opt-out informed consent procedure was implemented. Because it was neither feasible nor realistic 
to obtain informed consent from patients who had already been admitted to the ICU before the 
introduction of the opt-out procedure, permission was obtained from the BCWO to retrospectively 
include and anonymize patients, until the opt-out procedure became fully operational. Following 
BCWO approval, all newly admitted ICU patients or their legal representatives have been provided 
with an information leaflet about the study, included in the standard ICU admission folder. This allows 
them to decline participation at any time, in accordance with the approved opt-out procedure. 
 
8.4 Confidentiality of study data 
The investigator is responsible for treating study information as confidential. The investigator will 
ensure that the patient information will not be made publicly available. All patient study records are 
stored in a coded dataset to maintain the patients’ confidentiality. 
 
9 Administrative aspects 
9.1 Source data 
Source data are the medical records from MetaVision ® (iMDsoft, Tel Aviv, Israel) and Nexus used in 
Hospital Gelderse Vallei.  
 
9.2 Data handling 
The investigator is responsible for the design of the data collection forms. 
 
9.3 Documentation and storage 
All documents pertaining to the conduct of the study are kept by the investigator for a minimum 
period of 15 years in the digitalized archive system of the hospital. 
 
 
 

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