Effect of Intermittent Calorie Restriction on Metabolic Dysfunction-Associated Steatotic Liver Disease Patients With Abnormal Glucose Metabolism
Phase: NA
Conditions: Fatty Liver Disease, Type 2 Diabetes, Impaired Glucose Regulation
Sponsor: Shanghai Zhongshan Hospital

Effect of Intermittent Calorie Restriction on 
Metabolic Dysfunction-Associated Steatotic 
Liver Disease Patients with Abnormal 
Glucose Metabolism 
 
Department of Endocrinology and Metabolism,  
Zhongshan Hospital, Fudan University  
 
ClinicalTrials.gov ID: NCT04283942 
Date：2021-03-10 (Final version)  
 

Protocol  
 
1. Background 
Metabolic dysfunction-associated steatotic liver disease (MASLD) often coexists with type 
2 diabetes (T2DM) because of their common risk factors1. With the global epidemic trend of 
obesity and T2DM, MASLD has become an important cause of chronic liver disease in 
developed countries and rich areas in China. At present, the prevalence of MASLD in 
Chinese adults is 6-27%2-4. Compared with non-MASLD patients, the relative risk of type 2 
diabetes in patients with MASLD is 1.865. For patients with elevated blood glucose, the 
strategy of reducing liver fat content could also alleviate insulin resistance, contribute to better 
glucose control, and delay the occurrence of diabetes complications6. Due to the unclear 
pathogenesis, there is a lack of effective medication for MASLD. Lifestyle intervention is still 
the main treatment. Weight loss of 7-10% can effectively reduce liver steatosis and 
inflammation.  
Calorie restriction (CR) is an effective method to reduce weight and severity of obesity 
related diseases7,8, but traditionally it requires constant effort to cut down calorie intake every 
day (continuous calorie restriction, CCR) and may lead to potential adverse effects on the 
body. Intermittent calorie restriction (ICR) refers to maintaining no or very-low calorie intake 
periodically, which means alternating normal diet and fasting for a certain period to prevent 
and treat various diseases. The most common ICR schemes include time restrict fasting 
(TRF) or time restrict eating (TRE), alternate day fasting (ADF) and fasting for two days per 
week (5:2 diet). ICR can effectively reduce weight, lower blood pressure, improve lipid profile 
and reduce oxidative stress9-11. It also facilitates glucose control by increasing insulin 
sensitivity and improving β Cell function12,13. Recent studies have proved the effectiveness of 
TRE and ADF on reducing weight and improving cardiometabolic risk factors9,14-18. However, 
effect of ICR, especially 5:2 diet, on MASLD patients has been rarely studied. A few studies 
have found ICR could reduce liver enzymes in MASLD patients14,19,20, but they were less 
accurate because fat content of the whole liver, as well as of different liver segments were not 
quantitatively evaluated before and after treatment. More importantly, no study directly 
compared the therapeutic effect of ICR and CCR on MASLD currently.  
 
2. Aim of the study 
In the present study, we carried out a randomized, controlled, open-label clinical trial to 
investigate the efficacy and safety of ICR in MASLD patients with abnormal glucose metabolism. 
For ICR group, we adopt the 5:2 scheme which was related to stronger patient compliance12. 
The control group received traditional CCR. Two groups were instructed to keep overall calorie 
restriction to similar degree during 12 weeks of treatment. The changes of LFC of the whole 
liver, as well as of different liver segments measured by MRI-Proton Density Fat Fraction (MRI-
PDFF) were compared between two groups. We hypothesize that ICR will be more effective 
than CCR in reducing LFC and other metabolic risk factors. 
 
3. Research design 
3.1 Overall design 

This 12-week, open-label, randomized, controlled, parallel-group trial was conducted at 
Zhongshan Hospital Fudan University between July 2020 and July 2021 (ClinicalTrials.gov, 
NCT04283942). The trial was initiated by the researcher, performed in accordance with the 
ethical principles of the Declaration of Helsinki and approved by the Ethics Committee of 
Zhongshan Hospital Fudan University (approval number: B2019-256). A total of 60 patients 
were enrolled in the trial, and written informed consent was obtained from all patients. 
3.2 Randomization and masking 
The simple randomization method was used to ensure similar baseline characteristics 
between two groups. The randomization scheme will be provided by the Statistics Department 
of School of Public Health of Fudan University (generated by SAS software), and randomization 
will be completed by staff unrelated to this trial. According to the randomization scheme, a 
random envelope marked with the treatment number will be made and assigned for each 
subject according to the order of enrollment. The treatment will be carried out according to the 
arrangement of corresponding scheme. 
 
4. Study participants 
4.1 Inclusion Criteria 
• 
Age 18-70  
• 
Diagnosed as fatty liver by ultrasound or magnetic resonance imaging 
• 
BMI ≥ 24 kg/m2  
• 
Abnormal glucose metabolism: (meeting at least one):  
1) 
Impaired glucose regulation: fasting blood glucose ≥ 100 mg/dL, postprandial blood 
glucose ≥ 140 mg/dL or HbA1c ≥ 5.7% 
2) 
Diabetes mellitus: diabetic symptoms + plasma glucose concentration ≥ 200 mg/dL 
at any time or fasting plasma glucose concentration ≥ 100 mg/dL or OGTT 2 h plasma 
glucose concentration ≥ 200 mg/dL 
4.2 Exclusion Criteria 
• 
Type 1 diabetes, gestational diabetes and other special types of diabetes  
• 
Poor blood glucose control, HbA1c > 8.5% within 3 months  
• 
Taking antidiabetic drugs in the past month  
• 
Serum ALT > 6 times of normal upper limit 
• 
Excessive alcohol consumption (alcohol intake: men > 140 g, women > 70 g in the past 6 
months) 
• 
Other liver diseases: such as acute and chronic viral hepatitis, drug-induced hepatitis, 
immune hepatitis, liver cirrhosis, liver cancer, etc 
• 
Taking drugs that may affect MASLD in the past three months, such as vitamin E 
• 
Biliary obstructive diseases 
• 
Other diseases affecting glucose and lipid metabolism: hyperthyroidism, hypothyroidism, 
Cushing's syndrome, etc 
• 
Chronic kidney disease (serum creatinine ≥ 2.0 mg/dL) 
• 
Life expectancy of no more than 5 years 
• 
Already pregnant or plan to be pregnant in the near future 
• 
Mental illness 
• 
Other conditions affecting follow-up 

• 
Have participated in other clinical trials in the past 4 weeks 
• 
Absent of informed consent 
 
4.3 Termination criteria 
• 
Request to withdraw informed consents 
• 
Pregnancy 
• 
Systolic blood pressure ≥ 180 mmHg and diastolic blood pressure ≥ 110 mmHg 
• 
Liver enzymes (ALT or AST) increase to ≥ 6 times of the upper normal limit and last for 4 
weeks 
• 
Fasting blood glucose increases to > 200 mg/dL and lasts for 4 weeks 
• 
Serum creatinine increases to ≥ 1.5 mg/dL 
• 
During the treatment, drugs which may affect liver fat content are added or adjusted for 
dosage: such as hypoglycemic drugs, lipid-lowering drugs and liver protecting drugs 
 
5. Intervention 
After a 2-week screening phase, a total of 60 eligible patients will be randomized 1:1 into 
ICR and CCR group. 
Figure 1. Study design 
 
5.1 Dietetic intervention 
• 
ICR group: Participants in ICR group were instructed to ensure 2 successive days of 
fasting-mimicking and 5 days of recovery per week. On fasting-mimicking days, the 
participants were instructed to consume approximately 500 kcal/day and they were 
provided with plant-based meal replacement (ZhenBaiNian nutrition bar, Beijing Winlife 
Research Institute of Nutrition, Health, Food Science, and Technology, China) to improve 
adherence and ensured adequate intake of micronutrients. In the rest 5 days of recovery 
per week, participants were allowed to consume their usual diet. Main ingredients of this 
meal replacement are as follows: 
 
➢ 
Main ingredients: isomaltooligosaccharide solution, high-protein and high-fiber crispy 
grain (soy protein isolate, rice, polydextrose, calcium carbonate), Chia seed, resistant 
dextrin, coconut potato, salty vegetable grain, etc. 
 
Table 1. Information of nutrition bar used in this trial 
  
Per 100g 
%NRV 

energy 
355.4 kcal 
18% 
protein 
15.4 g 
26% 
Fat 
12.4 g 
21% 
Trans fats (acids) 
0 g 
 
Polyunsaturated fatty acids 
3.6 g 
 
carbohydrate 
34.0 g 
11% 
dietary fiber 
23.5 g 
94% 
sodium 
261 mg 
13% 
vitamin A 
565 μg  
71% 
vitamin D 
2.5 μg 
50% 
vitamin E 
8.05 mg 
58% 
Vitamin B1 
1.30 mg 
93% 
Vitamin B2 
1.30 mg 
93% 
Vitamin B6 
1.30 mg 
93% 
Vitamin B12 
0.80 μg 
33% 
vitamin C 
67.7 mg 
68% 
nicotinic acid 
13.50 mg 
96% 
folic acid 
328 μg  
82% 
Pantothenic acid 
3.17 mg 
63% 
Biotin 
3.8 μg 
13% 
phosphorus 
257 mg 
37% 
potassium 
318 mg 
16% 
magnesium 
72 mg 
24% 
calcium 
433 mg 
54% 
iron 
6.7 mg 
45% 
zinc 
5.25 mg 
35% 
selenium 
10.0 μg 
20% 
copper 
0.44 mg 
29% 
fluorine 
0.06 mg 
6% 
manganese 
1.00 mg 
33% 
ω~ 6 Linoleic acid 
1.6 g 
 
ω~ three α- linolenic acid 
2.0 g 
  
➢ 
Manufacturer: Beijing Wanlaikang Nutrition and Health Food Science and Technology 
Research Institute Co., Ltd 
➢ 
Product standard No.: Q/CPKWC 0085 
➢ 
Food production license No.: SC10611141312769 
 
• 
CCR group: Participants in CCR group were instructed to consume the prescribed calories 
(25 kcal / kg × [height (cm) – 100] kg21) every day by eating conventional food without time 
restriction. 
Both groups received dietary counseling from experienced nutritionists during the trial. 
Kitchen scales were distributed to participants, and they were encouraged to weigh food to 
ensure accurate reporting of their caloric intake. During the trial, all the participants were 
required to write daily dietary log. Using each log, nutritionists assessed their calorie intake 
each day based on the nutrient content shown on the Chinese Food Composition 

Table22.Participants received face-to-face instruction or app messages every week to assess 
their adherence to the prescribed diet.  
 
5.2 Physical activity 
During the trial, all participants were required to maintain their exercise routines and record 
their daily steps using a unified pedometer (Redmi Smart Band, Xiaomi Corporation, China). 
The use of drugs affecting blood glucose and fatty liver were avoided. 
 
6. Study outcomes 
6.1 Primary outcome 
• 
Change in liver fat content from baseline to 12 months 
6.2 Secondary outcomes 
• 
Change of weight  
• 
Change of blood glucose: fasting blood glucose,2h postload blood glucose, HbA1c  
• 
Change of liver enzymes: alanine aminotransferase, aspartate aminotransferase, γ-
glutamyl transpeptidase 
• 
Change of lipid profile: total cholesterol, triglyceride, high-density lipoprotein-cholesterol, 
low-density lipoprotein-cholesterol, free fatty acid  
6.3 Other pre-specified outcomes  
• 
Change of fat mass and lean mass measured by a bioimpedance analyzer 
• 
Change of abdominal adiposity measured by MRI: visceral adipose tissue and 
subcutaneous adipose tissue 
• 
Change of liver stiffness measured by liver transient elastography  
• 
Change of insulin sensitivity: fasting insulin and HOMA-IR (fasting glucose in mmol/L x 
fasting insulin in μU/mL)/22.5)  
 
7. Follow-up visits 
(1) Screening visit (V0): The purpose of this visit is to confirm subjects’ eligibility and interest 
in the proposed study, and obtain informed consent, relevant medical history (including 
diabetes, fatty liver, alcohol drinking history, hepatitis history, drug use, etc.), physical 
measurements (weight, height, waist circumference and blood pressure), and laboratory 
measurements (OGTT, HbA1c, liver and renal functions, electrolytes, hepatitis markers, 
etc). 1H-Proton Magnetic Resonance Spectroscopy (1H-MRS), MRI-Proton Density Fat 
Fraction (MRI-PDFF) and liver transient elastography were applied to evaluate the fatty 
liver. Body composition analysis and other examinations were performed. Relevant 
laboratory and imaging examination results within 2 weeks before screening can be 
approved.  
(2) Baseline visit (V1): The baseline follow-up was scheduled within 2 weeks after the 
screening period. Physical examination was conducted. Subjects will be required to record 
their diet and steps for 3 days before day 0 and the average of calorie consumption and 
steps will be used as baseline reference. Randomization will be conducted. 
(3) Follow up and termination visits (V2 - V9): At each visit, diet and step records will be 
collected. At week 2, 4, 8 12, physical measurements, fasting glucose and adverse events 
will be recorded. Nutritionists will provide dietary advice and help subjects to keep calorie 

restriction and a balance of nutrients. On this basis, liver and renal functions, electrolytes, 
fasting blood glucose and lipids profiles will be measured at week 4. At week 12 (termination 
visit), OGTT, 1H-MRS, MRI-PDFF, liver transient elastography and body composition 
analysis will be added. At week 1, 3, 6, 10, only telephone follow-up will be conducted. 
(4) Post-intervention visit (V10): At 4 weeks after the intervention (week 16), body weight will 
be investigated by telephone. 
 
Table 2. Study visits and data collection 
 
Screening Baseline 
 
 
 
 
 
 
 
Termin
ation 
Post-
interve
ntion 
Visit 
V0 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
Week 
<-2 
0 
1 
2 
3 
4 
6 
8 
10 
12 
16 
Day 
 
0 
7±3d  14±7d  21±3d  28±7d  42±3d  56±7d  70±3d  84±7d  112±3
d 
Inform consent 
Χ 
 
 
 
 
 
 
 
 
 
 
Randomization 
 
Χ 
 
 
 
 
 
 
 
 
 
Demographic 
Χ 
 
 
 
 
 
 
 
 
 
 
information 
History about fatty 
liver and 
hyperglycemia 
Χ 
 
 
 
 
 
 
 
 
 
 
Comorbidity and 
concomitant drug 
use 
Χ 
 
 
 
 
 
 
 
 
 
 
blood pressure 
Χ 
Χ 
 
Χ 
 
Χ 
 
Χ 
 
Χ 
 
Height 
Χ 
 
 
 
 
 
 
 
 
Χ 
 
Weight 
Χ 
Χ 
 
Χ 
 
Χ 
 
Χ 
 
Χ 
X 
Waist 
circumference 
Χ 
Χ 
 
Χ 
 
Χ 
 
Χ 
 
Χ 
 
Diet and steps 
records for 3 days 
 
Χ 
 
 
 
 
 
 
 
 
 
OGTT 
X 
 
 
 
 
 
 
 
 
Χ 
 
HbA1c 
Χ 
 
 
 
 
 
 
 
 
Χ 
 
liver and renal 
functions 
Χ 
 
 
 
 
Χ 
 
 
 
Χ 
 
Lipid profile 
Χ 
 
 
 
 
Χ 
 
 
 
Χ 
 
Fasting plasma 
glucose 
Χ 
 
 
Χ 
 
Χ 
 
Χ 
 
Χ 
 
Electrolytes 
Χ 
 
 
 
 
Χ 
 
 
 
Χ 
 
Routine blood test 
Χ 
 
 
 
 
Χ 
 
 
 
Χ 
 
Hepatitis markers 
and autoantibody 
Χ 
 
 
 
 
 
 
 
 
 
 
Urinalysis 
Χ 
 
 
 
 
 
 
 
 
Χ 
 
Liver transient 
elastography 
Χ 
 
 
 
 
 
 
 
 
Χ 
 
Liver 1H-MRS 
Χ 
 
 
 
 
 
 
 
 
Χ 
 
Liver MRI-PDFF 
Χ 
 
 
 
 
 
 
 
 
Χ 
 
Brain functional 
MRI 
Χ 
 
 
 
 
 
 
 
 
Χ 
 
Body composition 
Χ 
 
 
 
 
 
 
 
 
Χ 
 
Activity (steps) 
 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
 
Diet guidance 
 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
 
Blood sample 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Urine and feces 
sample 
 
X 
 
 
 
 
 
 
 
X 
 

Adverse events 
 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
Χ 
 
Distribute/Collect 
diet diary 
Χ 
Χ 
 
Χ 
 
Χ 
 
Χ 
 
Χ 
 
Distribute/Collect 
research food 
(only for ICR 
group) 
 
Χ 
 
Χ 
 
Χ 
 
Χ 
 
Χ 
 
 
8. Measurements and investigations 
Questionnaire: The research staff will apply a questionnaire obtaining information on history 
about fatty liver and hyperglycemia, comorbidity and concomitant drug use (such as fatty liver, 
diabetes, hypertension, dyslipidemia and cardiovascular disease), lifestyle risk factors 
(cigarette smoking, alcohol drinking and physical activity).  
Dietary record: The research staff will provide an electronic kitchen scale to subjects 
enrolled. Subjects are required to record the name and weight of food they eat in a whole day 
on a distributed form. After the diet record is finished, they should take a photo of form and send 
to dietitian in research group. The form is as followed. They are required to record food intake 
for 3 days before day 0 and the average of calorie consumption will be used as baseline 
reference. During intervention, subjects in both groups will get distributed blank forms in each 
visit and are required to record their diet every day. 
 
Table 3. Dietary form 
  
Date 
Breakfast 
Lunch 
Dinner 
Extra meals 
Item 
Food 
Weight 
(g) 
Food 
Weight 
(g) 
Food 
Weight 
(g) 
Food 
Weight 
(g) 
Staple 
food 
Rice 
 
  
  
  
  
  
  
  
Noodles 
 
  
  
  
  
  
  
  
Coarse 
grain 
 
  
  
  
  
  
  
  
Tubers 
 
  
  
  
  
  
  
  
Meat 
Pork 
 
  
  
  
  
  
  
  
Beef 
and 
mutton 
 
  
  
  
  
  
  
  
Chicken 
and 
duck 
 
  
  
  
  
  
  
  
Fish and 
shrimp 
 
  
  
  
  
  
  
  
Others 
 
  
  
  
  
  
  
  
Eggs 
  
  
  
  
  
  
  
  
Dairy food 
Milk 
  
  
  
  
  
  
  
  
Yogurt 
  
  
  
  
  
  
  
  
Others 
  
  
  
  
  
  
  
  
Bean 
products 
Bean 
curd 
  
  
  
  
  
  
  
  
Soybean 
milk 
  
  
  
  
  
  
  
  
Vegetables 
1 
  
  
  
  
  
  
  
  

2 
  
  
  
  
  
  
  
  
3 
  
  
  
  
  
  
  
  
4 
  
  
  
  
  
  
  
  
Fruits 
1 
  
  
  
  
  
  
  
  
2 
  
  
  
  
  
  
  
  
3 
  
  
  
  
  
  
  
  
Nuts 
  
  
  
  
  
  
  
  
Alcoholic 
drinks 
Beer 
  
  
  
  
  
  
  
  
Grape 
wine 
  
  
  
  
  
  
  
  
Millet 
wine 
  
  
  
  
  
  
  
  
Baijiu 
  
  
  
  
  
  
  
  
Non-alcoholic drinks 
  
  
  
  
  
  
  
  
Oils 
  
  
  
  
  
  
  
  
 
Activity (steps): During the intervention, subjects need to wear a smart electronic wearable 
device (Redmi Smart Band, Xiaomi Corporation, China) to record the daily steps automatically. 
Physical measurements: At the clinic visit, trained staff will take measurements of body 
weight, height, waist circumference, blood pressure according to standard procedures. 
Subjects should avoid wearing shoes and heavy clothes when measuring body weight. Mid-
way between the lower border of rib margin and the upper border of iliac crest will be measured 
as waist circumference. The blood pressure will be measured after resting for 5 minutes in a 
sitting position and alcohol, cigarettes, coffee/tea and physical exercise will be avoided within 
30 minutes before the measurement. Automatic sphygmomanometer will be applied for 
standardized detection. 
Laboratory measurements: Blood samples will be drawn 10 hours after fasting and be 
used to measure blood glucose, HbA1c, liver enzymes, serum creatinine, urine acid and lipids.. 
High performance liquid chromatography (HPLC) method will be used for measuring HbA1c.  
Total cholesterol, high-density lipoprotein cholesterol, triglycerides, free fatty acids, electrolytes, 
liver enzymes, serum creatinine and urine acid will be measured by a model 7600 automated 
bioanalyzer (Hitachi, Tokyo, Japan). Low-density lipoprotein cholesterol will be calculated by 
the Friedewald equation. The FBG and 2-hour postprandial blood glucose in oral glucose 
tolerance test will be tested following a standard 75g oral glucose tolerance test and measured 
using the glucose oxidase method. An electrochemiluminescence immunoassay will be used 
to measure the fasting and 2-hour postprandial insulin. All parameters are tested in the clinical 
biochemical laboratory of Zhongshan Hospital affiliated to Fudan University. The clinical testing 
laboratory will adopt the standardized process and obtain the corresponding certification 
qualification. 
1H-MRS: Whole liver fat content by 1H-MRS was performed with a 1.5T MRI Scanner 
(Siemens Healthineers, Germany). Firstly, T1-weighted sagittal, coronal, and axial images were 
acquired for the localization of the spectroscopy acquisition voxel. A single 2×2×2 cm voxel was 
placed within the right lobe avoiding major vascular structures and subcutaneous fat tissue. 
The proton spectrum was acquired after shimming over the volume of interest by means of a 
point-resolved spectroscopy sequence with the following parameters: repetition time = 1500 
ms and echo time = 135 ms. Signal intensities of the water peak at 4.8 ppm (Sw) and the fat 

peak at 1.4 ppm (Sf) were measured, and the hepatic fat percentage was calculated as a ratio 
of the signal from the methylene group to the total signal of methylene and water23. 
MRI-PDFF: MRI examinations were performed on a 3.0T MRI scanner (Ingenia CX 3.0 T, 
Philips Healthcare, USA)24-26. The quantitative calculation of fat fraction requires the use of MRI 
mDIXON-Quant_BH or FF sequences, because the dicom header information of these 
sequences already contains the value of fat fraction. However, the profile of the liver is not clear 
in these sequences, so we first use the deep learning neural network to predict the outline of 
the liver and the eight segments of the liver on the in-phase of dixon as it has a clear view of 
the liver. Then resample these two labels to the mDIXON-Quant_BH or FF sequence, and 
correspond to the location of each slice of these two sequences. In this way, the liver profile 
label and liver Couinaud segment label on the fat sequence are obtained. Afterwards, the 
corresponding matrix is obtained by multiplying the mDIXON-Quant_BH or FF sequence with 
the liver profile and liver Couinaud segment label matrix. Finally, the corresponding fat fraction 
mean and upper-lower percentiles of every liver segment can be calculated according to 
different needs. It should also be noted that the fat fraction values in different machines or 
sequences are in different units and need to be divided again by the corresponding ratios to 
obtain the final results. The neural network used for predicting liver profile and liver Couinaud 
segment is a 2.5D UNet. The input of this network is a multi-layer 2D matrix, and residual 
modules and channel attention mechanism modules are added on the basis of UNet. The 
training dataset Including more than 200 cases with manually corrected liver profile and liver 
Couinaud labels and from Quanjing Imaging Center and Zhongshan Hospital Affiliated to Fudan 
University. The ratio of training, verification and test datasets is 4:1:1, and the inference dice of 
the model test set is respectively 0.981 for liver profile and 0.804 for liver Couinaud segment. 
Transient elastography: The controlled attenuation parameter (CAP) in dB/m and Liver 
stiffness measurement (LSM) in kPa will be measured using FibroScan (FibroScan 502 Touch, 
Echosens, France)27. All subjects will be detected when holding breath in supine position by 
using the M probe. Each subject collected at least 10 valid measurements. Only when the 
proportion of effective measurement times to the total measurement times is ≥ 60%, the 
interquartile range (IQR) of repeatedly collected CAP value is ≤ 40 dB / m, and the IQR of 
repeatedly collected LSM value is ≤ 30%, can the result be considered reliable. The median 
value will be kept as a representative result. 
Body composition: Lean mass and fat mass were measured by a bioimpedance analyzer 
Inbody520 (Biospace, Seoul, Korea). Specifically, for the area of abdominal adipose tissue, 
MRI (1.5 T, Siemens Avanto, Erlangen, Germany) were applied to conduct abdominal 
positioning scanning first, and then 1H-MRS detection. Sections will be acquired through the 
abdomen at the level of umbilicus to L4. Subcutaneous adipose tissue (SAT) and visceral 
adipose tissue (VAT) will be measured with NIH image software (ImageJ 1.41o; National 
Institutes of Health, Bethesda, MD, USA)28. 
Blood samples in weeks 0 and week 12 were collected for metabolomics analysis. Feces 
samples in weeks 0 and week 12 were collected for detection of gut microbiome and gut 
microbiota-derived metabolites. Resting state cerebral functional magnetic resonance imaging 
was conducted before and after intervention to explore the effects of intervention on brain 
structure, network connectivity, and other parameters of brain functions. 
 

9. Adverse events (AE) 
9.1 Definition 
Non-Serious Adverse Event (NSAE) refers to an unforeseeable medical condition that 
occurs during or after the treatment, or an event that makes the original condition worse. The 
occurrence of NSAE does not necessarily have a causal relationship with the intervention. 
Adverse medical conditions include symptoms (such as nausea and chest pain), physical signs 
(such as tachycardia and hepatomegaly) or abnormal examination results. 
Serious adverse event (SAE) refers to the adverse event that meets one or more of the 
following criteria caused by intervention in screening method, treatment period, cleaning period 
or follow-up period: 
1) Causing death; 
2) Life-threatening injury; 
3) Leading to longer hospitalization; 
4) Causing continuous or obvious disability; 
5) Congenital malformation/defect; 
6) Major medical events that may cause injury to the subject or require medical intervention to 
prevent its occurrence. 
Severity grading of non-serious adverse events: 
1=mild (with symptoms or signs, but tolerable) 
2=moderate (feeling unwell and affecting normal activities) 
3=severe (inability to perform normal activities) 
The unresolved NSAE at the end of the trial need to be followed up. The follow-up interval 
and duration depend on the medical needs. For the unsolved adverse events at the end of the 
trial or when the subjects withdraw from the study, the follow-up shall be conducted until the 
NSAE returns to normal, or until the investigator believes that further follow-up is not necessary. 
9.2 Recording and assessment of adverse events 
Attention should be paid to the collection of AE during the whole research process. The 
following are the variables that should be recorded for each AE: onset and stop time, severity, 
measurements, causal relationship with intervention, outcomes and whether it is a serious 
adverse event. 
The severity of adverse events is assessed as follows: Mild: usually temporary, and not 
affecting daily activities; Moderate: causing discomfort and affecting normal activities, thought 
tolerable and not requiring participants to take any medication. Severe: disrupting daily activities 
and intolerable, requiring participants to take medication immediately. With regard to the causal-
effect relationship, medical adverse events are divided into five categories: definite, probable, 
possible, unlikely, and unrelated. Only “definite” and “probable” are counted as adverse events. 
The severity assessment of adverse events is as follows:  
1) 
Mild: usually temporary and does not affect daily activities. 
2) 
Moderate: discomfort, affect normal activity and does not require medication to alleviate 
symptoms. 
3) 
Serious: not endurable, affect daily activities and require medication immediately. 
9.3 Treatment of AE 
The patients with mild AE can continue the intervention and visits. Those with SAE should 
stop intervention and take corresponding treatment in time. 

Once NSAE happens, the severity, duration, treatment and outcome of adverse reactions 
should be explained. If other drugs are used at the same time during the trial, it shall be 
recorded in a timely manner on case report forms. 
All SAEs, whether related to the intervention or not, should be reported and recorded in 
the adverse event report form of the case report form, recording the occurrence time, severity, 
duration, measures taken, etc. Researchers have the responsibility to notify the Ethics 
Committee within 24 hours.  
 
10. Data management  
All case report forms for each participant should be filled out by study staff in a timely 
manner. The case report form should be double-checked for potential errors or missing data. 
All data, including questionnaires, physical examinations, laboratory and other examinations, 
will be filled in the subject’s chart. Original documents, participants’ charts, and CRF forms will 
be stored in the study office. All data will be double-entered by researcher staff.  
 
11. Statistics 
11.1 Statistical power and sample size 
A 12-week ICR has been reported to result in approximately 7% weight loss in 
overweight and obese participants, while the CCR group caused 30% less weight loss than 
the ICR group29. According to previous studies28,30, a 7% weight loss was approximately 
accompanied with a 19.3% reduction in LFC. Thus, a priori power calculation assumed 19.3% 
and 13.5% reduction in LFC in the ICR and CCR groups, respectively. Assuming an 
anticipated dropout rate of 15%, we estimated that an enrollment target of 60 participants (30 
per group) would provide the trial with 80% statistical power to detect a significant difference 
of 5.8±8% in absolute LFC change between ICR and CCR group at a significance level of 
0.05 using a two-tailed test.  
 
11.2 Data analysis 
Data were analyzed according to the intention-to-treat principle using R version 4.0.3. 
Results are presented as means ± SD unless otherwise noted. Paired t tests or Wilcoxon 
matched-pairs signed-ranks tests were used for comparison of continuous variables before 
and after 12-week intervention. While the chi-square or Fisher's exact tests were used for 
comparison of the categorical variables. Generalized linear models were used to compare the 
changes of the continuous variables after a 12-week intervention between the two groups, 
with adjustment for age, sex and baseline measures. All statistical analyses were two-sided, 
and P<0.05 was considered statistically significant. 
 
12. Quality control 
The trial will comply with China's Standard for Quality Management of Clinical Trials (GCP) 
and other laws and regulations related to clinical trials. The personal information of participants 
will be confidential and shall not be disclosed without permission. We will develop a 
standardized research procedure manual, including recruitment of subjects, form filling, 
intervention methods, parameter measurement methods, sample collection and management. 
All researchers will get relevant training before the start of the trial. 

 
13. Ethics approval 
This trial will comply with the Helsinki Declaration (1996 version) and Chinese clinical trial 
regulations. All amendments to the research protocol and informed consent will be approved 
by the Ethics Committee of Zhongshan Hospital affiliated to Fudan University. Adverse events 
must be reported to the Ethics Committee as required. The study will be registered on the NIH 
Clinical Trial website before enrollment. 
 
References 
1. 
Xia, M.F., Bian, H., and Gao, X. (2019). NAFLD and Diabetes: Two Sides of the Same Coin? 
Rationale for Gene-Based Personalized NAFLD Treatment. Frontiers in pharmacology 10, 
877. 10.3389/fphar.2019.00877. 
2. 
Fan, J.G., Kim, S.U., and Wong, V.W. (2017). New trends on obesity and NAFLD in Asia. 
Journal of hepatology 67, 862-873. 10.1016/j.jhep.2017.06.003. 
3. 
Fan, J.G. (2013). Epidemiology of alcoholic and nonalcoholic fatty liver disease in China. 
Journal of gastroenterology and hepatology 28 Suppl 1, 11-17. 10.1111/jgh.12036. 
4. 
Wang, F.S., Fan, J.G., Zhang, Z., Gao, B., and Wang, H.Y. (2014). The global burden of liver 
disease: the major impact of China. Hepatology (Baltimore, Md.) 60, 2099-2108. 
10.1002/hep.27406. 
5. 
Ballestri, S., Zona, S., Targher, G., Romagnoli, D., Baldelli, E., Nascimbeni, F., Roverato, A., 
Guaraldi, G., and Lonardo, A. (2016). Nonalcoholic fatty liver disease is associated with an 
almost twofold increased risk of incident type 2 diabetes and metabolic syndrome. 
Evidence from a systematic review and meta-analysis. Journal of gastroenterology and 
hepatology 31, 936-944. 10.1111/jgh.13264. 
6. 
Bril, F., and Cusi, K. (2016). Nonalcoholic Fatty Liver Disease: The New Complication of 
Type 2 Diabetes Mellitus. Endocrinology and metabolism clinics of North America 45, 
765-781. 10.1016/j.ecl.2016.06.005. 
7. 
Huang, M.A., Greenson, J.K., Chao, C., Anderson, L., Peterman, D., Jacobson, J., Emick, D., 
Lok, A.S., and Conjeevaram, H.S. (2005). One-year intense nutritional counseling results 
in histological improvement in patients with non-alcoholic steatohepatitis: a pilot study. 
The 
American 
journal of 
gastroenterology 
100, 
1072-1081. 
10.1111/j.1572-
0241.2005.41334.x. 
8. 
European Association for the Study of the, L., European Association for the Study of, D., 
and European Association for the Study of, O. (2016). EASL-EASD-EASO Clinical Practice 
Guidelines for the management of non-alcoholic fatty liver disease. Journal of hepatology 
64, 1388-1402. 10.1016/j.jhep.2015.11.004. 
9. 
Stekovic, S., Hofer, S.J., Tripolt, N., Aon, M.A., Royer, P., Pein, L., Stadler, J.T., Pendl, T., Prietl, 
B., Url, J., et al. (2020). Alternate Day Fasting Improves Physiological and Molecular 
Markers of Aging in Healthy, Non-obese Humans. Cell metabolism 31, 878-881. 
10.1016/j.cmet.2020.02.011. 
10. 
Saad, R. (2020). Effects of Intermittent Fasting on Health, Aging, and Disease. The New 
England journal of medicine 382, 1773. 10.1056/NEJMc2001176. 
11. 
Wei, M., Brandhorst, S., Shelehchi, M., Mirzaei, H., Cheng, C.W., Budniak, J., Groshen, S., 
Mack, W.J., Guen, E., Di Biase, S., et al. (2017). Fasting-mimicking diet and markers/risk 

factors for aging, diabetes, cancer, and cardiovascular disease. Science translational 
medicine 9. 10.1126/scitranslmed.aai8700. 
12. 
Sundfør, T.M., Svendsen, M., and Tonstad, S. (2018). Intermittent calorie restriction-a more 
effective approach to weight loss? The American journal of clinical nutrition 108, 909-910. 
10.1093/ajcn/nqy288. 
13. 
Cheng, C.W., Villani, V., Buono, R., Wei, M., Kumar, S., Yilmaz, O.H., Cohen, P., Sneddon, 
J.B., Perin, L., and Longo, V.D. (2017). Fasting-Mimicking Diet Promotes Ngn3-Driven β-
Cell Regeneration to Reverse Diabetes. Cell 168, 775-788.e712. 10.1016/j.cell.2017.01.040. 
14. 
Johari, M.I., Yusoff, K., Haron, J., Nadarajan, C., Ibrahim, K.N., Wong, M.S., Hafidz, M.I.A., 
Chua, B.E., Hamid, N., Arifin, W.N., et al. (2019). A Randomised Controlled Trial on the 
Effectiveness and Adherence of Modified Alternate-day Calorie Restriction in Improving 
Activity of Non-Alcoholic Fatty Liver Disease. Scientific reports 9, 11232. 10.1038/s41598-
019-47763-8. 
15. 
Liu, D., Huang, Y., Huang, C., Yang, S., Wei, X., Zhang, P., Guo, D., Lin, J., Xu, B., Li, C., et al. 
(2022). Calorie Restriction with or without Time-Restricted Eating in Weight Loss. The New 
England journal of medicine 386, 1495-1504. 10.1056/NEJMoa2114833. 
16. 
Trepanowski, J.F., Kroeger, C.M., Barnosky, A., Klempel, M.C., Bhutani, S., Hoddy, K.K., 
Gabel, K., Freels, S., Rigdon, J., Rood, J., et al. (2017). Effect of Alternate-Day Fasting on 
Weight Loss, Weight Maintenance, and Cardioprotection Among Metabolically Healthy 
Obese Adults: A Randomized Clinical Trial. JAMA internal medicine 177, 930-938. 
10.1001/jamainternmed.2017.0936. 
17. 
Jamshed, H., Steger, F.L., Bryan, D.R., Richman, J.S., Warriner, A.H., Hanick, C.J., Martin, C.K., 
Salvy, S.J., and Peterson, C.M. (2022). Effectiveness of Early Time-Restricted Eating for 
Weight Loss, Fat Loss, and Cardiometabolic Health in Adults With Obesity: A Randomized 
Clinical Trial. JAMA internal medicine 182, 953-962. 10.1001/jamainternmed.2022.3050. 
18. 
Wilkinson, M.J., Manoogian, E.N.C., Zadourian, A., Lo, H., Fakhouri, S., Shoghi, A., Wang, 
X., Fleischer, J.G., Navlakha, S., Panda, S., and Taub, P.R. (2020). Ten-Hour Time-Restricted 
Eating Reduces Weight, Blood Pressure, and Atherogenic Lipids in Patients with Metabolic 
Syndrome. Cell metabolism 31, 92-104.e105. 10.1016/j.cmet.2019.11.004. 
19. 
Mari, A., Khoury, T., Baker, M., Said Ahmad, H., Abu Baker, F., and Mahamid, M. (2021). 
The Impact of Ramadan Fasting on Fatty Liver Disease Severity: A Retrospective Case 
Control Study from Israel. The Israel Medical Association journal : IMAJ 23, 94-98. 
20. 
Wilson, R.B., Zhang, R., Chen, Y.J., Peters, K.M., Sawyez, C.G., Sutherland, B.G., Patel, K., 
Kennelly, J.P., Leonard, K.A., Jacobs, R.L., et al. (2020). Two-Week Isocaloric Time-
Restricted Feeding Decreases Liver Inflammation without Significant Weight Loss in Obese 
Mice with Non-Alcoholic Fatty Liver Disease. International journal of molecular sciences 
21. 10.3390/ijms21239156. 
21. 
Rössner, S. (2007). Paul Pierre Broca (1824-1880). Obesity reviews : an official journal of 
the International Association for the Study of Obesity 8, 277. 10.1111/j.1467-
789X.2007.00303.x. 
22. 
Y, Y. (2018). China food composition tables  
standard edition, 6th ed (Peking University Medical Press). 
23. 
Xia, M.F., Bian, H., Zhu, X.P., Yan, H.M., Chang, X.X., Zhang, L.S., Lin, H.D., Hu, X.Q., and 
Gao, X. (2018). Serum folic acid levels are associated with the presence and severity of 

liver steatosis in Chinese adults. Clin Nutr 37, 1752-1758. 10.1016/j.clnu.2017.06.021. 
24. 
Bonekamp, S., Tang, A., Mashhood, A., Wolfson, T., Changchien, C., Middleton, M.S., Clark, 
L., Gamst, A., Loomba, R., and Sirlin, C.B. (2014). Spatial distribution of MRI-Determined 
hepatic proton density fat fraction in adults with nonalcoholic fatty liver disease. Journal 
of magnetic resonance imaging : JMRI 39, 1525-1532. 10.1002/jmri.24321. 
25. 
Middleton, M.S., Heba, E.R., Hooker, C.A., Bashir, M.R., Fowler, K.J., Sandrasegaran, K., 
Brunt, E.M., Kleiner, D.E., Doo, E., Van Natta, M.L., et al. (2017). Agreement Between 
Magnetic Resonance Imaging Proton Density Fat Fraction Measurements and 
Pathologist-Assigned Steatosis Grades of Liver Biopsies From Adults With Nonalcoholic 
Steatohepatitis. Gastroenterology 153, 753-761. 10.1053/j.gastro.2017.06.005. 
26. 
de Padua, V.A.V., Dillman, J.R., and Trout, A.T. (2022). Retrospective comparison of liver 
chemical shift-encoded PDFF sampling strategies in children and adolescents. Abdom 
Radiol (NY) 47, 3478-3484. 10.1007/s00261-022-03615-0. 
27. 
Yang, X., Chang, X., Wu, S., Sun, X., Zhu, X., Wang, L., Xu, Y., Yao, X., Rao, S., Hu, X., et al. 
(2021). Performance of liver stiffness measurements obtained with FibroScan is affected 
by glucose metabolism in patients with nonalcoholic fatty liver disease. Lipids in health 
and disease 20, 27. 10.1186/s12944-021-01453-5. 
28. 
Liu, L., Yan, H., Xia, M., Zhao, L., Lv, M., Zhao, N., Rao, S., Yao, X., Wu, W., Pan, B., et al. 
(2020). Efficacy of exenatide and insulin glargine on nonalcoholic fatty liver disease in 
patients with type 2 diabetes. Diabetes/metabolism research and reviews 36, e3292. 
10.1002/dmrr.3292. 
29. 
Schübel, R., Nattenmüller, J., Sookthai, D., Nonnenmacher, T., Graf, M.E., Riedl, L., Schlett, 
C.L., von Stackelberg, O., Johnson, T., Nabers, D., et al. (2018). Effects of intermittent and 
continuous calorie restriction on body weight and metabolism over 50 wk: a randomized 
controlled 
trial. 
The 
American 
journal 
of 
clinical 
nutrition 
108, 
933-945. 
10.1093/ajcn/nqy196. 
30. 
Yan, H.M., Xia, M.F., Wang, Y., Chang, X.X., Yao, X.Z., Rao, S.X., Zeng, M.S., Tu, Y.F., Feng, 
R., Jia, W.P., et al. (2015). Efficacy of Berberine in Patients with Non-Alcoholic Fatty Liver 
Disease. PLoS One 10, e0134172. 10.1371/journal.pone.0134172.