Liver Fat as a Dietary Target of the Chinese Medical Nutrition Therapy (CMNT) Diet for Treating Type 2 Diabetes With Nonalcoholic Fatty Liver Disease
Phase: NA
Conditions: Type 2 Diabetes
Sponsor: State Key Laboratory of Subhealth Intervention Technology

Statistical Analysis Plan & Protocol 
for 
Liver fat as a dietary target by Chinese Medical Nutrition Therapy 
(CMNT) diet for treating type 2 diabetes with nonalcoholic fatty liver 
disease 
 
 
Protocol Approval 
CMNT Targeting Liver Fat in T2D with NAFLD 
 
Principal Investigator: 
Dongbo Liu, PhD, Professor 
State Key Laboratory of Subhealth Intervention Technology  
Horticulture College, Hunan Agricultural University 
No. 1 Nongda Road, Fu-Rong District, Changsha, 410128, P.R. China 
Tel.: +86-0731-8461-5293; Fax: +853-0731-8461-5293 
E-mail: liudongbo@hunau.net  
 
 
                             
 
Sponsor Name: State Key Laboratory of Subhealth Intervention Technology 
(Affiliated Institution: Hunan Agricultural University) 
 
                                
 
 
Date: February 12, 2025 

Signature Page 
Your signature on this page indicates that you confirm and agree to the statistical 
analysis of this trial in accordance with this plan. 
 
 
 
 
 
 
Project Leader's Approval Signature: ____________________ 
              
 
Date: __________________   
 
                         
 
 
Sponsor Name: State Key Laboratory of Subhealth Intervention Technology 
(Affiliated Institution: Hunan Agricultural University) 
 
 
 
Institutional Review Board (Signature, Seal):  
 
 
                          
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 Contents 
 
 
1. 
Original Statistical Analysis Plan for Version 2.0 ................................................................. 5 
1.1 Research Purpose ............................................................................................................ 5 
1.2  Sample Size Justification ............................................................................................... 5 
1.3 Outcome Analysis .............................................................................................................. 5 
2. 
The Statistical Plan-Amendments for Version 3.0 ................................................................ 6 
2.1 Research Purpose .............................................................................................................. 6 
2.2 Sample Size Justification .................................................................................................. 6 
2.3 Outcome Analysis .............................................................................................................. 6 
3. 
Statistical Analysis Plan Amendments. ................................................................................. 7 
3.1 Clarification of Data Analysis Set Definition .................................................................. 7 
3.2 Statistical Parameters ....................................................................................................... 7 
3.3 Addition of Interim Analysis ............................................................................................ 8 
4. 
Original study Protocol (Version 2.0) .................................................................................... 8 
4.1 Summary of Original Protocol： ..................................................................................... 8 
4.2 Research Population ......................................................................................................... 8 
4.3 Selection Criteria ............................................................................................................... 9 
4.3.1 Inclusion Criteria： ............................................................................................... 9 
4.3.2 Exclusion Criteria: ................................................................................................. 9 
4.4 Research Objectives ........................................................................................................ 10 
4.4.1 Primary Objective ................................................................................................ 10 
4.4.2 Secondary objective ............................................................................................. 10 
4.5 Outcome Measures .......................................................................................................... 11 
4.5.1 Primary Endpoints ............................................................................................... 11 
4.5.2 Secondary Endpoints ........................................................................................... 11 
4.6 Research Methods ........................................................................................................... 11 
4.7 Dietary Intervention Pattern .......................................................................................... 11 
4.8 Patient Recruitment ........................................................................................................ 12 
4.9 Research Procedures ....................................................................................................... 12 
4.10 Patient Follow-up .......................................................................................................... 13 
4.11 Efficacy and Safety Evaluation Monitored Adverse Effects/Toxicity ....................... 13 
4.12 Sources of Data for Research Subjects ........................................................................ 14 
4.13 Review of Patient Diabetes Management Data ........................................................... 15 
5. 
Study Protocol- Amendments (Version 3.0) ........................................................................ 15 
5.1 The Summary of Study Protocol .................................................................................... 15 
5.2 Research population ........................................................................................................ 15 
5.3 Selection Criteria ............................................................................................................. 16 
5.3.1 Inclusion Criteria: ................................................................................................ 16 
5.3.2 Exclusion Criteria: ............................................................................................... 16 
5.4 Research Objectives ........................................................................................................ 17 
5.4.1 Primary Objective ................................................................................................ 17 

5.4.2 Secondary Objectives ........................................................................................... 17 
5.5 Outcome Measures .......................................................................................................... 18 
5.5.1 Primary Endpoints ............................................................................................... 18 
5.5.2 Secondary Endpoints ........................................................................................... 18 
5.6 Study Method .................................................................................................................. 18 
5.7 Dietary Intervention Pattern .......................................................................................... 18 
5.8 Patient Recruitment ........................................................................................................ 19 
5.9 Research Procedures ....................................................................................................... 19 
5.10 Patient Follow-up .......................................................................................................... 20 
5.11 Safety Risk Assessment ................................................................................................. 20 
5.12 Data Sources .................................................................................................................. 21 
6  Study Protocol Amendments. ................................................................................................. 22 
 
 
 
 
 
 

1. Original Statistical Analysis Plan for Version 2.0 
 
1.1 Research Purpose 
This study aims to explore the efficacy of Chinese Medicine Nutrition Intervention 
(CMNT), an intermittent energy restriction (iER) intervention, in managing Chinese 
adults with Type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). It 
further explores whether the glucose-lowering effect of CMNT is related to the 
reduction of liver fat and its mechanism in hepatic glucose and lipid metabolism.  
 
 
1.2 Sample Size Justification 
Approximately 120 participants are expected to be enrolled across participating 
centers during the study period. The sample size calculation is based on the reduction 
in Glycated hemoglobin (HbA1c). According to preliminary studies, the mean and SD 
values of HbA1c after intervention were 7.173±1.919% in the CMNT group and 8.124
±1.144% in the control group. Forty-four participants in each group will be recruited, 
and the following formula will be used to calculate the required sample size. In 
consideration of stratification analysis on T2D and NAFLD comorbidity, we presume 
a dropout rate of 20%. In addition, as this study adds a 12-month follow-up compared 
to the control group's standard care, the sample size per group is increased to 60 cases. 
 
 
1.3 Outcome Analysis 
All statistical analyses will be conducted using IBM SPSS V.23. We will use a t-
test or χ2 test to determine the differences for normal data and Mann-Whitney U test for 
non-normal data between the CMNT group and usual care group. Data will be analyzed 
on an intention-to-treat basis, comparing the CMNT group with the usual care group. 
The controlled attenuation parameter (CAP) values and HbA1c values at baseline and 
after treatment will be evaluated by repeated measure analysis of variance. To control 
for confounders, the primary and secondary outcomes will be compared via analysis of 
covariance with age, sex, international physical activity questionnaire (IPAQ) score and 
adherence to the diet as covariates. Multivariate linear regression analysis will be 
performed to assess the association between changes in CAP values and changes in 
HbA1c values between two groups. P values < 0.05 are considered statistically 
significant and the data are analyzed using SPSS software 
A statistical analysis will be performed on all the enrolled patients who meet the 
protocol selection criteria and for whom physical examination indicators and fecal 
samples are available. Data will be summarized using standard descriptive statistics: 
-The continuous variables will be described in terms of data quantity, number of 
missing data, mean, SD, median, quartiles (Q1, Q3), minimum and maximum values. 
Means and standard deviations will be presented with one more decimal place than the 
collected data. 
-The categorical variables will be described in terms of missing data frequency, 
frequency and percentage of each recorded category. 

 
 
2. The Statistical Plan-Amendments for Version 3.0 
 
2.1 Research Purpose 
The main objective of this study is to investigate the effectiveness of CMNT (an 
intermittent fasting intermittent energy restriction intervention) in managing Chinese 
adults with T2D and NAFLD. It further explores whether the glucose-lowering effect 
of CMNT is related to reduced liver fat and its mechanism in hepatic glucose and lipid 
metabolism. This study is based on a small clinical pilot trial of 39 patients with type 2 
diabetes and non-alcoholic fatty liver disease and is a part of the CMNT study 
(NCT05439226). 
 
2.2 Sample Size Justification 
Approximately 120 participants are expected to be enrolled in the study. The 
sample size calculation is based on the reduction in HbA1c. According to preliminary 
studies, the mean ± SD values of HbA1c after intervention were 7.173%±1.919 in 
the CMNT group and 8.124% ±1.144 in the control group.29 Forty-four participants 
in each group will be recruited, and the following formula will be used to calculate the 
required sample size, with consideration of stratification analysis on T2D and NAFLD 
comorbidity, we presume a dropout rate of 20%. In addition, as this study adds a 12-
month follow-up compared to the control group's standard care, the sample size per 
group is increased to 60 cases. Reference for sample size calculation：Luo W, Zhou 
J, Yang X, et al. A Chinese medical nutrition therapy diet accompanied by intermittent 
energy restriction alleviates type 2 diabetes by enhancing pancreatic islet function and 
regulating gut microbiota composition. Food Res Int 2022;161:111744. 
An interim analysis will use sample data from the first 39 enrolled patients. The 
interim analysis sample size design is similar to that of the previous study (CMNT study: 
NCT05439226). The sample size estimation is based on the reduction in HbA1c. The 
estimated sample size of this trial is 24 per group, allowing a 20% dropout rate. 
However, due to the COVID-19 pandemic in China, we are only able to include 19-20 
subjects per group, which nevertheless proves sufficient to achieve > 80% statistical 
power. A subsequent analysis indicates the therapeutic potential of iER intervention in 
this study population. Reference for re-estimating the sample size calculation: Yang 
X, et al. Effect of an Intermittent Calorie-restricted Diet on Type 2 Diabetes Remission: 
A Randomized Controlled Trial. The Journal of Clinical Endocrinology & Metabolism 
108, 1415-1424 (2022). 
 
2.3 Outcome Analysis 
All statistical analyses will be conducted using IBM SPSS V.23. We will use a t-
test or χ2 test to determine the differences for normal data and Mann-Whitney U test 
for non-normal data between the CMNT group and usual care group. Data will be 
analyzed on an intention-to-treat basis, comparing the CMNT group with the usual care 
group. CAP values and HbA1C values at baseline and after treatment will be evaluated 

by repeated measure analysis of variance. To control the confounders, the primary and 
secondary outcomes will be compared via analysis of covariance with age, sex, IPAQ 
score and adherence to the diet as covariates. The multivariate linear regression analysis 
will be performed to assess the association between changes in CAP values and changes 
in HbA1c values between two groups. P values <0.05 are considered statistically 
significant and the data are analyzed using SPSS software. 
At baseline, the normality of continuous parameters will be tested. The test will be 
conducted in the modified Intention-to-Treat Analysis (ITT) population. If two or more 
of the Shapiro-Wilk, Cramer-von Mises, and Anderson-Darling tests are statistically 
significant at the 0.05 level, the normality of the distribution will be visually inspected. 
If necessary, simple transformations (e.g., taking the natural logarithm) can be applied 
to parameters to normalize the distribution. 
Unless otherwise specified, the two groups will be made as follows: 
- Continuous parameters: compared using t tests; if the normality of the distribution 
cannot be assumed, the non-parametric Wilcoxon test will be used. 
- Categorical parameters: compared using the chi-square test; if the theoretical 
frequency is less than 5, Fisher's exact test or an equivalent method will be used. 
Statistical analysis will be performed on all enrolled patients who meet the 
protocol selection criteria and for whom physical examination indicators and fecal 
samples are available. Data will be summarized using standard descriptive statistics: 
- Continuous variables will be described by data quantity, number of missing data, mean 
± standard deviation (SD), median, quartiles (25%, 75%), minimum and maximum 
values. 
- Categorical (ordinal or nominal) variables will be described by missing data frequency, 
frequency and percentage of each recorded category. 
 
 
 
3. Statistical Analysis Plan Amendments. 
The main revisions and their justifications are as follows: 
3.1 Clarification of Data Analysis Set Definition 
In the final statistical plan, the modified ITT population is defined as all enrolled 
participants with CAP data at baseline, replacing the Full Analysis Set (FAS) in the 
original plan. 
Rationale: To more precisely identify assessable participants directly related to the core 
research objectives, enhancing the clinical significance and reliability of the results. 
 
3.2 Statistical Parameters 
In the final statistical plan, tests for the normality of continuous parameters have been 
added, along with the specific methods for comparing the two groups. 
Rationale: Adding normality tests for continuous parameters and specifying group-
comparison methods in the final plan ensures the applicability of the statistical methods 
and the scientific rigor of the results. 
 

3.3 Addition of Interim Analysis 
Rationale: To evaluate the effectiveness of the CMNT dietary intervention and the 
safety of the energy-restricted diet during clinical trials, we conducted a mid-term 
analysis when 40% of the intended treatment population was reached. The study 
subjects were individuals with type 2 diabetes who had non-alcoholic fatty liver disease 
(NAFLD). It remains unknown whether their outcomes and risks differ from those of 
conventional care. Notably, due to differences in age, biochemical profiles, and the 
severity of underlying conditions, each participant's tolerance to the energy restricted 
diet varied. The purpose of the interim analysis was to assess the effectiveness of the 
CMNT dietary intervention, as well as to evaluate participants' adherence and the safety 
of the energy-restricted diet. Based on previous research, a smaller sample size of 24 
participants per group was deemed statistically significant. Therefore, the first 40% of 
the participants were selected for the interim analysis. 
 
References: Yang X, et al. Effect of intermittent calorie-restricted diet on the remission 
of type 2 diabetes: a randomized controlled trial. Journal of Clinical Endocrinology & 
Metabolism 108,1415-1424 (2022).  
 
 
 
4. Original study Protocol (Version 2.0) 
4.1 Summary of Original Protocol： 
Principal Sponsor: State Key Laboratory of Subhealth Intervention Technology 
(Affiliated Institution: Hunan Agricultural University)  
Title: Chinese Medicine Nutrition Therapy Targeting Liver Fat for the Treatment of 
Type 2 Diabetes with Non-Alcoholic Fatty Liver Disease  
Abbreviation: CMNT Targeting Liver Fat in T2D with NAFLD 
Principal Investigator (PI):  
Dongbo Liu (Professor) 
College of Horticulture, Hunan Agricultural University 
State Key Laboratory of Subhealth Intervention Technology  
Number of Research Centers:  
Hunan Shanshui physical examination center； 
Physical Examination Center of Gezhouba central hospital of Sinopharm 
Jinheyuan Outpatient Department, National Sub-health Intervention Technology 
Achievement Application Center 
Timeline: Recruitment Phase: June 5, 2022 to May 5, 2024 (anticipated)  
Follow-up Phase: 1 years  
Maximum Study Duration: 5 years 
 
4.2 Research Population 
-Patients officially diagnosed with T2D and NAFLD. 
-Patients included in those using antidiabetic medications and newly-diagnosed patients 
not using such medications.  

-All patients have undergone a risk assessment for iER intervention in multi-
disciplinary team meetings (including endocrinologists, nutritionists, and researchers) 
and have been evaluated by physicians for the need for appropriate clinical medications.  
-Patients included in the study undergo diagnostic examinations (imaging or biological) 
in conjunction with diet and drug therapy (if any), in accordance with the 
recommendations of the Chinese health authorities. 
 
 
4.3 Selection Criteria 
4.3.1 Inclusion Criteria： 
-Patients with T2DM diagnosed according to the 1999 WHO diagnostic criteria for 
diabetes and with fatty liver, diagnosed according to Chinese standards.  
- BMI at screening between 18.0 and 35.0 kg/m2 (inclusive); age at screening between 
18 and 75 years (inclusive), both males and females;  
- Willing and able to accurately self-monitor blood glucose using a home glucose meter; 
-Able to understand and comply with the trial procedures, voluntarily participate in the 
trial, and provide informed consent. 
 
4.3.2 Exclusion Criteria: 
-Patients with type 1 diabetes, pancreatic - related diabetes, or secondary diabetes (e.g., 
diabetes caused by Cushing's syndrome or acromegaly). 
-Use of any of the following medications or treatments: 
-Use of other medications that may affect glucose metabolism within the past 2 months, 
including systemic glucocorticoids (excluding inhaled or topical use), growth hormones, 
etc.; 
-Use of antihypertensive or lipid-lowering medications that have not reached a stable 
dosage before screening; 
-Presence of any of the following medical histories or conditions: 
-History or presence of any cardiac disease within the past 6 months; 
-Decompensated heart failure (NYHA Class III or IV); 
-Unstable angina, myocardial infarction, coronary artery bypass grafting, or coronary 
stenting; 
-Uncontrolled or severe arrhythmias (e.g., long QT syndrome), as assessed by the 
investigator as unsuitable for trial participation; 
-History of hemorrhagic or ischemic stroke within the past 6 months, as assessed by the 
investigator as unsuitable for trial participation; 
-Medical history of cerebral thrombosis, cerebral vascular blockage, encephalic 
angioma, transient ischemic attack, cerebral hemorrhage, stroke, hydrocephalus, or 
malignant brain tumor;  
-History of carotid artery stenting; 
-Urinary system conditions such as nephrotic syndrome, uremia, polycystic kidney 
disease, kidney transplantation, unilateral nephrectomy/congenital solitary kidney, 
renal atrophy, renal tumor; 
-Digestive system conditions such as ascites, liver cirrhosis, liver fluke infection, severe 

hepatitis, gastric varices; 
-Nervous system conditions such as cerebellar atrophy, demyelinating diseases, 
cerebral palsy, Parkinson's disease, mania, schizophrenia; 
-Respiratory system conditions such as pulmonary embolism, cor pulmonale; 
-Musculoskeletal system conditions such as arterial rupture, myeloma; 
-Immune system conditions such as Behçet's disease, lupus erythematosus; 
-Conditions such as chondrosarcoma, liposarcoma, brucellosis, leukemia; 
-History of malignant tumor within the past 5 years, or currently under evaluation for 
potential malignancy; 
-History of unstable or treatment-requiring proliferative retinopathy or macular edema 
within the past 6 months; 
-History of diabetic ketoacidosis or hyperosmolar nonketotic diabetic coma within the 
past 6 months; 
-Currently suffering from lower extremity arteriosclerotic occlusive disease; 
-History of severe infection or severe trauma within the past 1 month; 
-History of ≥2 severe hypoglycemic episodes within the past year; 
-Currently suffering from clinically significant urinary tract/genital infection, or a 
history of complicated urinary tract infections, or recurrent urinary tract infections 
within the past 6 months; 
-Currently suffering from uncontrolled hypertension, defined as systolic blood pressure 
≥160 mmHg and/or diastolic blood pressure ≥100 mmHg at screening/baseline, or 
systolic blood pressure ≤90 mmHg and/or diastolic blood pressure ≤60 mmHg; 
-Currently suffering from uncontrolled thyroid dysfunction; 
-History of other severe endocrine diseases, such as multiple endocrine neoplasia; 
-History of severe hepatic or renal disease; 
-Suspected or confirmed history of alcohol or drug abuse; 
-Blood donation or blood loss ≥400 mL within the past 3 months; 
-Presence of severe psychiatric disorders or language barriers, unwillingness or 
inability to fully understand and cooperate; 
-Pregnant or breastfeeding women; 
-Any other conditions deemed unsuitable for trial participation by the investigator. 
 
 
4.4 Research Objectives 
4.4.1 Primary Objective 
The effect of CMNT intervention on liver fat and blood glucose in patients with T2D 
and NAFLD 
 
4.4.2 Secondary objective 
- Analyze whether blood glucose improvement is related to liver fat reduction.  
- Changes in liver function. 
- Changes in blood lipid levels.  
- Changes in systemic inflammatory levels.  
- Improvement in insulin resistance for this comorbidity.  

- Changes in liver fibrosis.  
- Changes in gut microbiota. 
 
4.5 Outcome Measures 
4.5.1 Primary Endpoints 
Liver fat content measured by transient elastography 
Glycated hemoglobin (HbA1c) 
 
4.5.2 Secondary Endpoints 
Anthropometric measures: height, weight, waist circumference, hip circumference, 
chest circumference, pulse, blood pressure, heart rate, body temperature, respiratory 
rate; 
Body fat composition analysis;  
Complete blood count;  
Fasting blood glucose; Fasting insulin;  
Fasting C-peptide;  
12-item liver function tests: total protein, albumin, globulin, albumin/globulin ratio, 
total bilirubin, direct bilirubin, indirect bilirubin, ALT, AST, GGT, ALP, AST/ALT; 7-
item lipid profile: triglycerides, total cholesterol, HDL, LDL, apolipoprotein A, 
apolipoprotein B, apolipoprotein A/apolipoprotein B ratio;  
High-sensitivity C-reactive protein (hsCRP);  
β-hydroxybutyrate;  
Insulin - like growth factor -1 (IGF-1);  
Free fatty acids;  
Food frequency questionnaire (FFQ);  
Gut microbiota. 
 
4.6 Research Methods 
This is a multi-center, prospective, longitudinal study to be jointly conducted at 
three clinical recruitment centers in China: Hunan Shanshui Health Check-up Center; 
Sinopharm Gezhouba Central Hospital; and the National Sub - health Intervention 
Technology Application Center (Jinhe Garden Clinic). 
 
4.7 Dietary Intervention Pattern 

 
CMNT Intervention Group: The CMNT diet adopts a "5 + 10" intermittent energy 
restriction pattern, with a 15 - day CMNT cycle consisting of 5 consecutive days of 
CMNT diet followed by 10 days of normal diet for participants in the trial group. During 
the 5-day CMNT diet period, participants in the CMNT group receive a pre-prepared 
CMNT diet totaling 917 Kcal daily. Specifically, breakfast includes a packet (50g) of 
fruit and vegetable porridge; lunch consists of a packet (60g) of nutritional rice and a 
cup (25g) of solid beverage; dinner comprises two blocks (30g) of meal-replacement 
biscuits and a cup of solid beverage. The 10-day normal diet follows the standards of 
the China Type 2 Diabetes Prevention and Control Guidelines. The control group 
adheres to the conventional diet as per China Type 2 Diabetes Prevention and Control 
Guidelines.  
 
4.8 Patient Recruitment 
During dietary intervention and routine diabetes management counseling, 
researchers will inform patients of the possibility to participate in the study. Patients 
will receive both verbal and written information on the study protocol, primary 
objectives, implementation, follow - up, and sample collection (as part of the study 
protocol). All sample collections will coincide with routine clinical examinations 
during conventional treatment. Researchers will distribute information leaflets to 
patients and collect signed informed consent forms. 
 
4.9 Research Procedures 
All procedures outlined in the study protocol will be conducted concurrently with 
routine clinical examinations in conventional treatment. Procedures specific to this 
study include: 
Collection of one blood and one fecal sample at three time points: 
-At recruitment (Baseline). 
-Post-treatment (90 days). 
-During follow-up (6, 12, and 24 months). 
- High-frequency blood glucose monitoring using a glucose meter, with data uploaded 
to the CMNT mobile app for timely access by nutritionists and endocrinologists. 
- Electronic collection of patient - completed responses to quality-of-life and food 
questionnaires. These questionnaires will be distributed in paper form during the 

baseline physical examination. 
- Electronic collection of patient-completed responses to the ethnic background 
questionnaire. This questionnaire will only be requested after an additional informed 
consent form is signed. 
 
In accordance with the "Administrative Measures for Ethical Review of Life Sciences 
and Medical Research Involving Human Beings" jointly issued by the National Health 
Commission, Ministry of Education, Ministry of Science and Technology, and the 
National Administration of Traditional Chinese Medicine, this study is considered 
research involving human subjects. 
 
4.10 Patient Follow-up 
Each patient will be followed up according to the conventional diabetes 
management pathway, including: 
- Follow-ups at 6, 12, and 24 months post - intervention. 
- Systematic follow-up or telephone contact within one year to assess disease status. 
- The data collection methods for this study refer to the six major data collection 
methods summarized in "Research Methods, Design, and Analysis" by Larry 
Christensen and Burke Johnson. The collected data are health-related and are deemed 
appropriate, relevant, and not excessive for the research purposes.  
The following data will be collected for each patient participating in the study: 
- Identification information: 
- The identification number consists of the center number in the study, a sequential 
number increasing according to the order of recruitment (with the first number in the 
study being 1), and the first two letters of the surname and the initial of the given name. 
- The aforementioned inclusion and exclusion criteria. 
- Data strictly necessary for conducting the research: 
-Age. 
-Clinical examination. 
-Laboratory tests. 
-Results of specific biomarker analyses in blood samples related to liver function. 
-Medical imaging (transient elastography). 
-Results of gut microbiota detection in fecal samples. 
-Data on any adverse events and/or side effects occurring during the study period. 
-Personal or family medical history. 
-Dates related to the study (especially the recruitment date and visit dates). 
-Family status. 
-Socio - occupational category/occupation. 
-Participation in other studies or research to ensure compliance with inclusion criteria. 
-Tobacco, alcohol, and drug use. 
-The quality-of-life and dietary questionnaires. 
 
4.11 Efficacy and Safety Evaluation Monitored Adverse Effects/Toxicity 
All risks and discomforts associated with calorie-restricted diets include hunger, 

anxiety, somnolence, dizziness, headache, muscle pain, fatigue, low blood pressure, and 
in rare cases, fainting. These dietary interventions may also lead to arrhythmias, short-
term nutritional deficiencies, and weakened immune responses. For individuals already 
malnourished, long-term low-calorie diets are particularly hazardous. Participants may 
withdraw from the dietary intervention and resume their normal diet at any time during 
the study. If significant discomfort occurs during the study, participants should seek 
immediate medical attention. During the dietary intervention, participants should drink 
sufficient water to prevent dehydration and avoid intense activities or exercises. They 
should refrain from operating motor vehicles and heavy machinery, avoid high-
temperature environments such as hot showers or baths, and abstain from alcohol. If 
participants have any questions about the dietary intervention or feel unwell, they 
should contact the researcher, consult their personal physician, or seek immediate 
medical care. After the dietary intervention, participants should avoid overeating and 
gradually return to a normal diet, starting with liquid foods like soups and juices, then 
gradually transitioning to light meals. Participants may feel dizzy during blood 
collection. In rare cases, participants may experience bruising, excessive bleeding, 
infection (a minor risk when the skin is punctured), dizziness, and fainting. Participants 
may stop the blood collection procedure at any time. If bleeding or infection occurs 
during blood collection, participants should contact their personal physician or seek 
immediate medical care. 
Participants may withdraw from the dietary intervention and resume their normal 
diet at any time during the study. If participants experience significant discomfort 
during the study, they should seek immediate medical attention. 
 
Adverse Event Reporting: Procedures for Reporting Unexpected and Fatal Toxicities 
within 10 working days of becoming aware of the event, the following serious adverse 
events (SAEs) or serious suspected adverse reactions will be reported to the IRB: 
- Death; 
- Hospitalization; 
- Persistent or significant disability; 
- Severe impact on the ability to perform normal life activities; 
- Events endangering the participant; or 
- Events potentially requiring medical or surgical intervention to prevent any of the 
above - listed outcomes. 
 
4.12 Sources of Data for Research Subjects 
-Responses to the quality of life and food questionnaires provided by research 
participants. In this research, these questionnaires will be distributed in paper form. 
-Data related to diabetes management and the study, provided by researchers: results of 
clinical examinations, laboratory tests, medical imaging (transient elastography), data 
on adverse events and side effects occurring during the study period, personal or family 
medical history.  
Researchers will input all this data into an electronic case report form from the patient's 
medical records. 

-Results of gut microbiota detection in fecal samples analyzed by a sequencing 
company. 
All collected data will be anonymized and stored securely. 
 
 
4.13 Review of Patient Diabetes Management Data 
All anonymized diabetes management data included in the study (mainly routine 
physical examination and imaging data) will be reviewed by medical experts to ensure 
correct classification. Data reviewers will remain blinded to the results of fecal sample 
sequencing. If the medical reviewer identifies any inconsistencies between the data 
reported by the centers in the study and the review results, clarification will be requested. 
 
 
5. Study Protocol- Amendments (Version 3.0) 
5.1 The Summary of Study Protocol 
Principal Sponsor: State Key Laboratory of Subhealth Intervention Technology 
(Affiliated Institution: Hunan Agricultural University)  
Title: Chinese Medicine Nutrition Therapy Targeting Liver Fat for the Treatment of 
Type 2 Diabetes with Non-Alcoholic Fatty Liver Disease  
Abbreviation: CMNT Targeting Liver Fat in T2D with NAFLD 
Principal Investigator (PI):  
Dongbo Liu (Professor) 
College of Horticulture, Hunan Agricultural University 
State Key Laboratory of Subhealth Intervention Technology  
Number of Research Centers:  
Hunan Shanshui physical examination center； 
Physical Examination Center of Gezhouba central hospital of Sinopharm; 
Jinheyuan Outpatient Department, National Sub-health Intervention Technology 
Achievement Application Center; 
Yiyang Central Hospital (Hunan Province, China); 
Shaoyang Central Hospital (Hunan Province, China); 
Zhuzhou People's Hospital (Hunan Province, China); 
Timeline: Recruitment Phase: June 5, 2022 to May 5, 2025 (anticipated)  
Follow-up Phase: 1 years  
Maximum Study Duration: 5 years 
 
5.2 Research population 
-Patients officially diagnosed with T2D and NAFLD.  
-The study includes patients using antidiabetic medications and newly-diagnosed 
patients not using such medications.  
-All patients have undergone a risk assessment for intermittent energy restriction diet 
intervention in multidisciplinary team meetings (including endocrinologists, 
nutritionists, and researchers) and have been evaluated by physicians for the need for 
appropriate clinical medications.  

-Patents included in the study undergo diagnostic examinations (imaging or biological) 
in conjunction with diet and drug therapy (if any), in accordance with the 
recommendations of the Chinese health authorities. 
 
5.3 Selection Criteria 
5.3.1 Inclusion Criteria: 
- Patients with type 2 diabetes mellitus diagnosed according to the 1999 WHO 
diagnostic criteria for diabetes and with fatty liver, diagnosed according to Chinese 
standards. 
- BMI at screening greater than 18.0 kg/m2; age at screening between 18 and 75 years 
(inclusive), both males and females; 
- Willing and able to accurately self - monitor blood glucose using a home glucose 
meter;  
- Be able to understand and comply with the trial procedures, voluntarily participate in 
the trial, and provide informed consent. 
 
 
5.3.2 Exclusion Criteria: 
-Patients with type 1 diabetes, pancreatic - related diabetes, or secondary diabetes (e.g., 
diabetes caused by Cushing's syndrome or acromegaly). 
-Use of any of the following medications or treatments: 
-Use of other medications that may affect glucose metabolism within the past 2 months, 
including systemic glucocorticoids (excluding inhaled or topical use), growth hormones, 
etc.; 
-Use of antihypertensive or lipid-lowering medications that have not reached a stable 
dosage before screening; 
-Presence of any of the following medical histories or conditions: 
-History or presence of any cardiac disease within the past 6 months; 
-Decompensated heart failure (NYHA Class III or IV); 
-Unstable angina, myocardial infarction, coronary artery bypass grafting, or coronary 
stenting; 
-Uncontrolled or severe arrhythmias (e.g., long QT syndrome), as assessed by the 
investigator as unsuitable for trial participation; 
-History of hemorrhagic or ischemic stroke within the past 6 months, as assessed by the 
investigator as unsuitable for trial participation; 
-Medical history of cerebral thrombosis, cerebral vascular blockage, encephalic 
angioma, transient ischemic attack, cerebral hemorrhage, stroke, hydrocephalus, or 
malignant brain tumor;  
-History of carotid artery stenting; 
-Urinary system conditions such as nephrotic syndrome, uremia, polycystic kidney 
disease, kidney transplantation, unilateral nephrectomy/congenital solitary kidney, 
renal atrophy, renal tumor; 
-Digestive system conditions such as ascites, liver cirrhosis, liver fluke infection, severe 
hepatitis, gastric varices; 

-Nervous system conditions such as cerebellar atrophy, demyelinating diseases, 
cerebral palsy, Parkinson's disease, mania, schizophrenia; 
-Respiratory system conditions such as pulmonary embolism, cor pulmonale; 
-Musculoskeletal system conditions such as arterial rupture, myeloma; 
-Immune system conditions such as Behçet's disease, lupus erythematosus; 
-Conditions such as chondrosarcoma, liposarcoma, brucellosis, leukemia; 
-History of malignant tumor within the past 5 years, or currently under evaluation for 
potential malignancy; 
-History of unstable or treatment-requiring proliferative retinopathy or macular edema 
within the past 6 months; 
-History of diabetic ketoacidosis or hyperosmolar nonketotic diabetic coma within the 
past 6 months; 
-Currently suffering from lower extremity arteriosclerotic occlusive disease; 
-History of severe infection or severe trauma within the past 1 month; 
-History of ≥2 severe hypoglycemic episodes within the past year; 
-Currently suffering from clinically significant urinary tract/genital infection, or a 
history of complicated urinary tract infections, or recurrent urinary tract infections 
within the past 6 months; 
-Currently suffering from uncontrolled hypertension, defined as systolic blood pressure 
≥160 mmHg and/or diastolic blood pressure ≥100 mmHg at screening/baseline, or 
systolic blood pressure ≤90 mmHg and/or diastolic blood pressure ≤60 mmHg; 
-Currently suffering from uncontrolled thyroid dysfunction; 
-History of other severe endocrine diseases, such as multiple endocrine neoplasia; 
-History of severe hepatic or renal disease; 
-Suspected or confirmed history of alcohol or drug abuse; 
-Blood donation or blood loss ≥400 mL within the past 3 months; 
-Presence of severe psychiatric disorders or language barriers, unwillingness or 
inability to fully understand and cooperate; 
-Pregnant or breastfeeding women; 
-Any other conditions deemed unsuitable for trial participation by the investigator. 
 
5.4 Research Objectives 
5.4.1 Primary Objective 
The effect of CMNT intervention on liver fat and blood glucose in patients with 
T2D and non-alcoholic fatty liver disease. 
 
5.4.2 Secondary Objectives 
- Analyze whether blood glucose improvement is strongly related to liver fat reduction. 
- Changes in liver function. 
- Changes in blood lipid levels.  
- Changes in systemic inflammatory levels.  
- Improvement in insulin resistance for this comorbidity. 
- Changes in liver fibrosis. 
- Changes in gut microbiota.  

- Use metabolomics to identify key metabolites.  
- Based on gut microbiota findings, screen for key microbial taxa post-CMNT 
intervention, and validate the causal relationship between diet and gut microbiota using 
fecal microbiota transplantation. 
 
 
5.5 Outcome Measures 
5.5.1 Primary Endpoints 
-Liver fat content measured by transient elastography 
-Glycated hemoglobin (HbA1c) 
 
5.5.2 Secondary Endpoints 
-Anthropometric measures: height, weight, waist circumference, hip circumference, 
chest circumference, pulse, blood pressure, heart rate, body temperature, respiratory 
rate;  
-Body fat composition analysis; 
-Complete blood count;  
-Fasting blood glucose; Fasting insulin;  
-Fasting C-peptide;  
-Liver function tests: total protein, albumin, globulin, albumin/globulin ratio, total 
bilirubin, direct bilirubin, indirect bilirubin, ALT, AST, GGT, ALP, AST/ALT;  
-Lipid profile: triglycerides, total cholesterol, HDL, LDL, apolipoprotein A, 
apolipoprotein B, apolipoprotein A/apolipoprotein B ratio;  
-High - sensitivity C-reactive protein (hCRP); 
-β-hydroxybutyrate;  
-Insulin-like growth factor-1 (IGF-1);  
-Free fatty acids; 
-Food frequency questionnaire (FFQ);  
-Gut microbiota;  
-Untargeted metabolomics;  
-Fecal and single-strain transplantation. 
 
 
5.6 Study Method 
This is a multi-center, prospective, longitudinal study initially conducted at three 
clinical recruitment centers: Hunan Shanshui Health Check-up Center; Sinopharm 
Gezhouba Central Hospital; and the National Sub-health Intervention Technology 
Application Center (Jinhe Garden Clinic). The study will be expanded to six research 
centers, with new additions signing letters of intent with the undertaking organization. 
The Interim Analysis: In the 120-participant CMNT intervention project, interim 
analyses will be added to compare intervention effects before and after CMNT diet 
optimization and with the conventional control group. 
 
5.7 Dietary Intervention Pattern 

 
CMNT Intervention Group: The CMNT diet adopts a "5 + 10" intermittent energy 
restriction pattern, with a 15-day CMNT cycle consisting of 5 consecutive days of 
CMNT diet followed by 10 days of normal diet for participants in the trial group. During 
the 5-day CMNT diet period, participants in the CMNT group receive a pre-prepared 
CMNT diet totaling 917 Kcal daily. Specifically, breakfast includes a packet (50g) of 
fruit and vegetable porridge; lunch consists of a packet (60g) of nutritional rice and a 
cup (25g) of solid beverage; dinner comprises two blocks (30g) of meal-replacement 
biscuits and a cup of solid beverage. The 10-day normal diet follows the standards of 
the China Type 2 Diabetes Prevention and Control Guidelines. The control group 
adheres to the conventional diet as per China Type 2 Diabetes Prevention and Control 
Guidelines.  
 
5.8 Patient Recruitment 
During dietary intervention and routine diabetes management counseling, 
researchers will inform patients of the possibility to participate in the study. Patients 
will receive both verbal and written information on the study protocol, primary 
objectives, implementation, follow-up, and sample collection (as part of the study 
protocol). All sample collections will coincide with routine clinical examinations 
during conventional treatment. Researchers will distribute information leaflets to 
patients and collect signed informed consent forms. 
 
5.9 Research Procedures 
All procedures outlined in the study protocol will be conducted concurrently with 
routine clinical examinations in conventional treatment. Procedures specific to this 
study include: 
- Collection of one blood and one fecal sample at three time points: 
-At recruitment (baseline). 
-Post-treatment (90 days). 
-During follow - up (6, 12, and 24 months). 
- High-frequency blood glucose monitoring using a glucose meter, with data uploaded 
to the CMNT mobile app for timely access by nutritionists and endocrinologists. 
-Electronic collection of patient-completed responses to the quality-of-life and food 
questionnaires. These questionnaires will be distributed in paper form during the 
baseline physical examination. 
- Electronic collection of patient-completed responses to the ethnic background 

questionnaire. This questionnaire will only be requested after an additional informed 
consent form is signed. 
In accordance with the "Administrative Measures for Ethical Review of Life 
Sciences and Medical Research Involving Human Beings" jointly issued by the 
National Health Commission, Ministry of Education, Ministry of Science and 
Technology, and the National Administration of Traditional Chinese Medicine, this 
study is considered research involving human subjects. 
 
5.10 Patient Follow-up 
Each patient will be followed up according to the conventional diabetes 
management pathway, including: 
-Follow-ups at 6, 12, and 24 months post-intervention. 
-Systematic follow - up or telephone contact within one year to assess disease status. 
The data collection methods for this study refer to the six major data collection 
methods summarized in "Research Methods, Design, and Analysis" by Larry 
Christensen and Burke Johnson. The collected data are health-related and are deemed 
appropriate, relevant, and not excessive for the research purposes.  
The following data will be collected for each patient participating in the study: 
 
-Identification information: 
-The identification number consists of the center number in the study, a sequential 
number increasing according to the order of recruitment (with the first number in the 
study being 1), and the first two letters of the surname and the initial of the given name. 
-The aforementioned inclusion and exclusion criteria. 
-Data strictly necessary for conducting the research: 
-Age. 
-Clinical examination. 
-Laboratory tests. 
-Results of specific biomarker analyses in blood samples related to liver function. 
-Medical imaging (transient elastography). 
-Results of gut microbiota detection in fecal samples. 
-Data on any adverse events and/or side effects occurring during the study period. 
-Personal or family medical history. 
-Dates related to the study (especially the recruitment date and visit dates). 
-Family status. 
-Socio-occupational category/occupation. 
-Participation in other studies or research to ensure compliance with inclusion criteria. 
-Tobacco, alcohol, and drug use. 
-The quality of life and dietary questionnaires. 
 
5.11 Safety Risk Assessment 
Risks and discomforts associated with calorie - restricted diets include hunger, 
anxiety, somnolence, dizziness, headache, muscle pain, fatigue, low blood pressure, and 
in rare cases, fainting. These dietary interventions may also lead to arrhythmias, short-

term nutritional deficiencies, and weakened immune responses. For individuals already 
malnourished, long-term low-calorie diets are particularly hazardous. Participants may 
withdraw from the dietary intervention and resume their normal diet at any time during 
the study. If significant discomfort occurs during the study, participants should seek 
immediate medical attention. During the dietary intervention, participants should drink 
sufficient water to prevent dehydration and avoid intense activities or exercises. They 
should refrain from operating motor vehicles and heavy machinery, avoid high-
temperature environments such as hot showers or baths, and abstain from alcohol. If 
participants have any questions about the dietary intervention or feel unwell, they 
should contact the researcher, consult their personal physician, or seek immediate 
medical care. After the dietary intervention, participants should avoid overeating and 
gradually return to a normal diet, starting with liquid foods like soups and juices, then 
gradually transitioning to light meals. Participants may feel dizzy during blood 
collection. In rare cases, participants may experience bruising, excessive bleeding, 
infection (a minor risk when the skin is punctured), dizziness, and fainting. Participants 
may stop the blood collection procedure at any time. If bleeding or infection occurs 
during blood collection, participants should contact their personal physician or seek 
immediate medical care. 
Participants may withdraw from the dietary intervention and resume their normal 
diet at any time during the study. If participants experience significant discomfort 
during the study, they should seek immediate medical attention. 
 
Adverse Event Reporting: Procedures for Reporting Accidental and Fatal 
Toxicities  
Within 10 working days of becoming aware of the event, the following serious adverse 
events (SAEs) or serious suspected adverse reactions will be reported to the IRB: 
- Death; 
- Hospitalization; 
- Persistent or significant disability; 
-Severe impact on the ability to perform normal life activities; 
- Events endangering the participant; or 
- Events potentially requiring medical or surgical intervention to prevent any of the 
above the listed outcomes. 
 
 
5.12 Data Sources 
-Responses to the quality-of-life and food questionnaires provided by study participants. 
In this study, these questionnaires will be distributed in paper form. 
-Data related to diabetes management and the study, provided by researchers: results of 
clinical examinations, laboratory tests, medical imaging (transient elastography), data 
on adverse events and side effects occurring during the study period, personal or family 
medical history. Researchers will input all this data into an electronic case report form 
from the patient's medical records. 
-Results of gut microbiota detection in fecal samples analyzed by a sequencing 

company. 
-Results of fecal and single-strain transplantation validation. 
All collected data will be anonymized and stored securely. 
 
5.13 Review of Patient Diabetes Management Data 
All anonymized diabetes management data included in the study (mainly routine 
physical examination and imaging data) will be reviewed by medical experts to ensure 
correct classification. Data reviewers will remain blinded to the results of fecal sample 
sequencing. 
If the medical reviewer identifies any inconsistencies between the data reported 
by the centers in the study and the review results, clarification will be requested. 
 
 
 
 
6  Study Protocol Amendments. 
The main revisions and their justifications are as follows: 
6.1 Addition of Interim Analysis. 
Rationale: The purpose of adding an interim analysis is to evaluate the effectiveness of 
the CMNT dietary intervention during this clinical trial, while also assessing participant 
compliance and the safety of the energy-restricted diet. The baseline CMNT 
intervention follows a "5+10" intermittent energy restriction structure (also referred to 
as iER, intermittent Energy Restriction), with the first 5 days using a fixed 917 kcal/day 
meal replacement model. Our prior research has shown that this baseline CMNT dietary 
structure is superior to conventional care in glycemic control for type 2 diabetes (T2D) 
management, with more participants achieving diabetes remission. However, this study 
focuses on T2D patients comorbid with non-alcoholic fatty liver disease (NAFLD), and 
whether its efficacy and risks differ from conventional care remains unknown. Notably, 
individual responses to energy restriction vary due to differences in age, metabolic 
characteristics, and baseline disease severity. For some participants, this fixed dietary 
model may not be the optimal intervention. Therefore, we have incorporated an interim 
analysis to preliminarily assess the efficacy and potential risks of the baseline CMNT 
diet in this comorbid population. Based on the unblinded interim data, the Data and 
Safety Monitoring Board (DSMB) will vote on whether to continue the original CMNT 
dietary protocol, using the following predefined criteria: 
-Continue the original CMNT protocol if it demonstrates significantly better efficacy 
in glycemic and lipid control compared to conventional care, with no additional safety 
risks. 
- Optimize the dietary protocol if efficacy is equivalent or inferior to conventional care, 
provided no safety concerns exist. 
-Terminate the dietary protocol if safety risks are significantly higher than conventional 
care, even if efficacy is observed. 
 

6.2 Expansion of BMI Inclusion Criteria. 
The BMI inclusion criterion was revised from "18-35" to " > 18" (removing the upper 
limit) to include patients with higher BMI values. 
Rationale: In clinical practice, some patients with a BMI over 35 have shown interest 
in participating in this study. After evaluation by physicians, these patients can be 
included. 
 
6.3 Increase in the Number of Multicenter Sites 
The number of centers increased from 3 to 6, including multiple municipal medical 
institutions (Yiyang Central Hospital, Shaoyang Central Hospital, Zhuzhou People's 
Hospital). 
Rationale: Expanding the number of multicenter sites accelerates participant 
recruitment. Given the study's focus on patients with T2D and NAFLD, the recruitment 
of patients with comorbidities is relatively challenging. Additionally, expanding the 
number of centers enhances the generalizability of the study's conclusions, making 
them applicable to patients in more regions. 
 
6.4 Clarification and Revision of Secondary Objectives and Corresponding 
Evaluation Criteria.     
-Addition of metabolomics content to identify key metabolites in CMNT intervention. 
-Screening for key microbial taxa post - CMNT intervention based on gut microbiota 
results. 
-Validation of the causal relationship between diet and gut microbiota using fecal 
transplantation. 
-These secondary objectives give rise to the following outcome measures: untargeted 
metabolomics, fecal and single - strain transplantation trials. 
Rationale: Incorporating gut microbiota and metabolomics indicators helps better 
explore the mechanism of CMNT dietary intervention in this population. 
 
6.5 The participant recruitment period has been extended to May 5, 2026. 
Rationale: Due to the severe COVID-19 pandemic during 2022-2023, coupled with the 
fact that this study focuses on comorbidities, the number of eligible participants has 
been significantly lower compared to studies involving general T2D patients. 
Furthermore, as most individuals currently do not prioritize fatty liver screening, 
participant recruitment has progressed more slowly than anticipated. Therefore, an 
extension of the recruitment period is necessary. 
 
All revisions have been approved by the Ethics Committee.