A Randomized Controlled Trial on the Effectiveness and Adherence of Modified Alternate-day Calorie Restriction (MACR) in Improving Activity of Non-Alcoholic Fatty Liver Disease
Phase: NA
Conditions: Non-Alcoholic Fatty Liver Disease
Sponsor: Universiti Sains Malaysia

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Research Protocol 
 
Protocol title 
A Randomized Controlled Trial on the Effectiveness and Adherence of Modified Alternate-
day Calorie Restriction (MACR) in Improving Activity of Non-Alcoholic Fatty Liver Disease 
 
 
Sponsor 
Universiti Sains Malaysia, Health Campus, 16150, Kubang Kerian, Kota Bharu, Kelantan, 
Malaysia 
- 
USM short term grant (reference no: 304/PPSP/61313173) 
- 
Research University Individual (RUI) grant (reference no: 1001/PPSP/812151) 
 
 
Investigators 
Dr Muhammad-Izzad Johari1,A,B,E,F, Prof Lee Yeong Yeh1,A,E,G, Dr Zheng Feei Ma1,C,D, Dr 
Juhara Haron1,B, Dr Chandran Nadarajan1,B, Dr Khairiah Mat Yusoff1,B,F, Dr Wan Nor 
Ariffin1,C,D, Mdm Khairun Nisah Ibrahim2,B, Mdm Nurhazwani Hamid1,C,D, SN Bee Eng 
Chua3,B 
 
1School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu Malaysia 
2Nutrition & Dietetic Unit, Universiti Sains Malaysia, Kota Bharu, Malaysia 
3Hospital Universiti Sains Malaysia, Kota Bharu, Malaysia 
 
 
AStudy Design 
BData Collection 
CStatistical Analysis 
DData Interpretation 
EManuscript Preparation 
FLiterature Search 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PROJECT SUMMARY 
Rationale: There is no effective therapy for non-alcoholic fatty liver disease (NAFLD), 
although intensive calorie restriction is typically recommended but dietary adherence is an 
issue. The current study aimed to determine the effectiveness and adherence of eight 
weeks of modified intermittent fasting in the control of NAFLD activity.  
Objective: Primary objective are to determine the effectiveness of 8 weeks of MACR in 
changes of BMI, biochemical and ultrasound parameters in NAFLD. The secondary objective 
is to evaluate the adherence rate of MACR in NAFLD patients. 
Method: This is a randomized, single-blind controlled trial with 8-weeks modified alternate-
day calorie restriction (MACR) as the active intervention and normal habitual diet as control 
in Hospital Universiti Sains Malaysia with study duration for 24 months. The outcome 
measures are included changes in BMI, blood lipids, fasting blood sugar, liver enzymes and 
ultrasonographic measurements of liver steatosis and shear wave elastography (SWE). Per-
protocol (PP) and intention-to-treat (ITT) analysis are performed within and between-groups 
with P < 0.05 as significant.  
Expected outcome: Specifically, we expected here that MACR regimen resulted in a mean 
weight loss between 3-5% from baseline after 8 week of intervention thus it is also expected 
to decreases in several key biomarkers for CAD risk, such as total cholesterol, LDL 
cholesterol, triacylglycerols and blood pressure. Liver parameter which included liver 
enzymes, steatosis and fibrosis would also expect to improve after 8 weeks MACR. 
Additionally, a similar adherence rate is expected to achieve during the MACR regime when 
compared with other alternate day calorie restriction regime. 
Funding: USM short term grant (reference no: 304/PPSP/61313173) and Research 
University Individual (RUI) grant (reference no: 1001/PPSP/812151). 
Keywords 
Non-alcoholic fatty liver disease; fatty liver; calorie restriction; liver steatosis; liver fibrosis 
 
 
 
 
 
 
 
 
 
 
 
 

Rationale & background information 
 
Disease activity and progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic 
steatohepatitis (NASH) and cirrhosis can be highly variable (1), where 2-3% will eventually 
progress to end-stage liver diseases. With the rising prevalence of metabolic syndrome and 
obesity, NAFLD has become the most frequent form of chronic liver disease in the West but 
also in Asia (2, 3). 
 
There are good evidence that weight loss is effective in improving liver histology in NAFLD, 
for example, Pomrat et al randomised 31 obese patients with NASH into intensive lifestyle 
changes over 48 weeks versus structured basic education only, and the intensive lifestyle 
group showed significant improvements in steatosis, necrosis, and inflammation (4). Intense 
calorie restriction is the recommended form of dietary strategy for management of NAFLD. 
Even though such intense dietary strategy has proven to be effective (4, 5), some patients 
find it difficult to adhere and maintain.  
 
On the other hand, intermittent fasting achieves more consistent weight loss by improving 
adherence (6, 7), as intermittent fasting only requires calorie restriction every other day 
compared to conventional form of daily calorie restriction (8). Alternate day calorie 
restriction can be divided into two components, a ‘feed day’ and a ‘fast day’ where food is 
consumed ad libitum for 24 hours period alternating with either complete or partial 
(modified) calorie restriction for the next 24 hours. Modified alternate-day calorie 
restriction (MACR), the dietary strategy employed in our study, restricts 70% of an 
individual’s daily requirement of calorie per day (8). There are other forms of intermittent 
fasting, for example, 2-4 days of ad libitum feeding alternating with 2-4 days of calorie 
restriction (8-11).   
 
Currently, there are no approved pharmacological therapies for NAFLD, and many guidelines 
advocate recommendation with a focus on controlling risk factors and lifestyle interventions 
that include dietary and physical activities. No specific NAFLD trials have evaluated the 
effectiveness of modified form of intermittent fasting in the control of NAFLD activity. 
Therefore, our study aimed to determine the effectiveness of 8 weeks of MACR in changes 
of BMI, biochemical and ultrasound parameters in NAFLD and to evaluate the adherence 
rate of such dietary strategy. 
 
References 
 
1. Angulo P, Hui J, Marchesini G, et al. The NAFLD fibrosis score: a noninvasive system 
that identifies liver fibrosis in patients with NAFLD. Hepatology 2007;45:847e54. 
 
2. Alberti KG, Eckel RH, Grundy SM et al. Harmonizing the metabolic syndrome: a joint 
interim statement of the International Diabetes Federation Task Force on 
Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American 
Heart Association; World Heart Federation; International Atherosclerosis Society; 
and International Association for the Study of Obesity. Circulation 2009;120:1640–5. 
 
3. Almeda-Valdes P, Cuevas-Ramos D, Aguilar-Salinas CA. Metabolic syndrome and non-
alcoholic fatty liver disease. Ann Hepatol. 2009;8(Suppl. 1):S18–24. 
 

4. Promrat K, Kleiner DE, Niemeier HM, Jackvony E, Kearns M, Wands JR, et al. 
Randomized controlled trial testing the effects of weight loss on nonalcoholic 
steatohepatitis. Hepatology 2010; 51:121-129. 
 
5. Harrison SA, Oliver D, Arnold HL, et al. Development and validation of a simple 
NAFLD clinical scoring system for identifying patients without advanced disease. Gut 
2008; 57:1441e7. 
 
6. Malik VS, Hu FB. Popular weight-loss diets: from evidence to practice. Nat Clin Pract 
Cardiovasc Med. 2007; 4:34–41. 
 
7. Das SK, Gilhooly CH, Golden JK, et al. Long-term effects of 2 energy-restricted diets 
differing in glycemic load on dietary adherence, body composition, and metabolism 
in CALERIE: a 1-y randomized controlled trial. Am J Clin Nutr 2007; 85:1023–30. 
 
8. Varady KA, Hellerstein MK. Alternate-day fasting and chronic disease prevention: a 
review of human and animal trials. Am J Clin Nutr 2007; 86: 7–13. 
 
9. Li Y, Liu L, Wang B, Wang J, Chen D. Metformin in nonalcoholic fatty liver disease: a 
systematic review and meta-analysis. Biomed Rep 2013;1:57-64 
 
10. Musso G, Gambino R, Cassader M, Pagano G. A metaanalysis of randomized trials for 
the treatment of nonalcoholic fatty liver disease. HEPATOLOGY 2010;52:79-104. 159 
 
11. Soccio RE, Chen ER, Lazar MA. Thiazolidinediones and the promise of insulin 
sensitization in type 2 diabetes. Cell Metab 2014;20:573-591. 
 
12. Omer Z, Cetinkalp S, Akyildiz M, Yilmaz F, Batur Y, Yilmaz C, et al. Efficacy of insulin 
sensitizing agents in nonalcoholic fatty liver disease. Eur J Gastroenterol Hepatol 
2010;22:18-23. 
 
13. Dhumal Uttareshvar Mahaling, Madole Mahesh Basavaraj, Aher Jagdish Bika. 
Comparison of lipid profile in different grades of non-alcoholic fatty liver disease 
diagnosed on ultrasound. Asian Pac J Trop Biomed. 2013 Nov; 3(11): 907 
 
14.  National Center for Health Statistics. Third National Health and Nutrition 
Examination Survey: Hepatic Steatosis Assessment Procedure Manual. Hyattsville, 
MD: National Center for Health Statistics.; 2010. 
 
 
Study goals and objectives 
 
Research question 
1. Does of MACR associated with improvement of mean liver steatosis and 
elastrography using shear wave elastography in NAFLD patients? 
2. Does MACR associated with improvement of biochemical parameter (lipid profile, 
liver chemistries) in NAFLD patients? 
3. Does MACR have a good adherence rate for dietary strategy 
 
 
 

Study goals 
To determine the effectiveness of 8 weeks of MACR in changes of BMI, biochemical and 
ultrasound parameters in NAFLD and to evaluate the adherence rate of such dietary 
strategy. 
 
Primary objective  
To determine the effectiveness of 8 weeks of MACR in changes of BMI, biochemical and 
ultrasound parameters in NAFLD  
 
Secondary objective 
To evaluate the adherence rate of MACR in NAFLD patients 
 
Study Design 
 
Type of study 
Randomized control trial 
 
Study Duration and location 
Study Location              :  Gastroenterology Clinic and Radiology Department Hospital USM. 
Study Duration 
 :  24 months 
 
Sampling Population and sample 
Reference Population: 
o 
 Non-Alcoholic Fatty Liver Disease (NAFLD) 
 
Source Population 
Gastroenterology Clinic in Hospital Universiti Sains Malaysia (HUSM), Kota Bharu, Kelantan 
NAFLD patient 
 
Sampling Frame 
Inclusion criteria  
  
1.  Age ranges 18 to 70 years old              
2.  Baselines Ultrasound show evidence of fatty liver 
3.  Elevated alanine aminotransferase (ALT) or aspartate aminotransferase (AST)   
     values (ALT>41 or AST>34U/L) 
                
Exclusion criteria 
1. Medically or surgically ill patients who cannot consent  
2. Complicated chronic liver disease with portal hypertension 
3. Significant alcohol consumption (1 standard drink per day) 
4. Contraindications to calorie restriction 
5. Pregnancy 
6. Engagement in an active weight loss program 
7. Taking weight-loss medication, substance abuse 
8. Significant psychiatric problems 
 
Withdrawal criteria 
1. Subject who unable to tolerate alternate daily fasting intervention during the 
trial 
2. Patient own choice 
 

Methodology  
 
Participants 
NAFLD patients who attending the Gastroenterology Clinic at Hospital Universiti Sains 
Malaysia, a tertiary referral centre for the north-eastern Peninsular Malaysia, will be screen 
from August 2015 till July 2016. 
 
During the first visit, if participants met the selection criteria, informed consent are then 
taken. Demographic data of participants are recorded. All participants are required to 
complete a two-week run-in period consisting of maintaining and self-monitoring their usual 
dietary and daily activities.   
 
Participants who fulfilled the selection criteria are randomly assign to the MACR group or 
the control group after a 2 week run-in period. During the 2 weeks, participants are 
requiring to keep their body weight stable by maintaining their usual eating habits and their 
daily activities.  Randomization is performed using a random number generator with 
recruitment aimed for three MACR participants for every control participant by principal 
investigator. Calculated sample size of 46 participants would achieve a significant difference 
in studied outcome between intervention and control group using a two-sided test with α = 
0.05 and power = 0.8. Participants are blinded after assignment to interventions or control 
group. 
 
Interventions 
 
MACR group  
For a total of 8 weeks, participants in this group need to restrict 70% of their energy needs 
over 24 hours on a calorie restriction day alternate with a feeding day for the next 24 hours, 
where participants allowed eating whatever they wish (ad libitum). The calorie restriction 
and feeding days begin at 9am each day, and on the calorie restriction day, meals are 
consumed between 2pm and 8pm to ensure that each participant underwent the same 
duration of calorie restriction. The MACR protocol is shown in Figure 1. On each calorie 
restriction day, participants are allowed to take energy-free beverages, tea, coffee, and 
sugar-free gum and encouraged to drink plenty of water. Diet plans are not provided to 
participants but are self-selected using detailed individualized food portion lists, meal plans, 
and recipes. To ensure maximal adherence to dietary plan, participants received phone calls 
from the investigator (Izzad) and 2-weekly appointments (total four appointments) with a 
dietician described below. Adverse experiences are assessed every 2 weeks until 1 week 
after the completion of trial. 
 
 
 
 
 
 
 
 
 
 
 
 
 

Figure 1 MACR protocol for the 8 weeks duration 
 
 
 
Assessment of dietary plan adherence 
 
A designated dietician (KhairunNisah) will provide education to participants regarding 
calorie restriction and help to monitor their dietary compliance. Adherence to intervention 
is assessed via a diet diary and dietary recall. The diary is collected and reviewed by the 
dietician every 2 weeks. If the diary indicates that the participants eat an extra calorie on a 
fasting day, that day is labelled as “not adherent.” If the diary revealed that the participant 
did not eat any extra food item, that day is labelled as “adherent.” Adherence data are 
assessed each week as 1) absolute adherence (number of adherent days with diet) and 2) 
percentage of adherence calculated by applying the following formula for adherence in 
percentage. The formula is shown below. 
 
 
Number of fast day’s adherent 
_________________________       X   100 
Number of fast days in week  
 
 
Control group  
 
All participants in the control group will continue their usual habitual diet for 8 weeks. No 
specific dietary advice or educations are provided throughout the entire trial. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Calorie restriction day  
Feeding day  
Calorie restriction day 
No restriction  
(ad-libitum) 
Start: 9am 
End: 9am  
(on next day) 
70% calorie restriction  
Start: 9am 
Meal: 2pm & 8pm 
End: 9am  
(on next day) 
70% calorie restriction   
Start: 9am  
Meal: 2pm & 8pm  
End: 9am 
(on next day) 

Figure 2 Flow Diagram 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Management of diabetes medications during the study period  
 
Previous reports found that Insulin sensitizing agents like thiazolidinediones and metformin 
might induce biochemical and histological effects similar with NAFLD (9-12). Therefore, 
participants, who were already taking thiazolidinedione or metformin, must be on a stable 
regimen for at least 6 months prior to this study enrolment and the regimen must remain 
stable during the study.  All participants are not allowed to start on any of these 
medications during the entire study period to avoid any potential confounding effects. 
Participants who continue to have active disease despite on these medications should be 
allowed to participate in the study trial to maximize generalizability. If medically necessary, 
participants were allowed new medications for hyperglycaemia and the available options 
included sulfonylureas, meglitinides and insulin. 
 
Assessed for eligibility (n=) 
Excluded (n=) 
Not meeting inclusion criteria (n=) 
Declined to participate (n=) 
Other reason (n=) 
Analysed  
Excluded from analysis (reason) (n=) 
2-weekly follow up till completion of trial 
Lost to follow-up (n=) 
Discontinued intervention (n=) 
Allocated to 8-weeks MACR group (n=) 
Received allocated intervention (n=)  
Did not receive allocated intervention (n=) 
At 1-week after completion of trial 
Lost to follow-up (n=) 
Discontinued intervention (n=) 
Allocated to Usual Habitual Diet group (n=) 
 Received allocated intervention (n=) 
 Did not receive allocated intervention (n=) 
Analysed  
Excluded from analysis (reason) (n=) 
Allocation 
Analysis 
Follow-Up 
Randomized (n=) 
Enrolments 

Procedures 
 
A standardized calibrated balance (Secca, ZT-120, and Hamburg, Germany) will be used to 
measure weight of participants in light clothing at each visit. A wall-mounted stadiometer 
(Secca, ZT-120, Hamburg, Germany) is used to measure height (in m), and together with 
weight (in Kg), body mass index (BMI) is calculated. Waist circumferences are measured 
with the participants standing, and with the measuring tape kept horizontal and measured 
at a level midway between the superior aspect of the iliac crests and the lower lateral 
margins of the ribs. 
 
Blood samples - 8-10 hours of fasting blood samples are collected at 8-10 am at baseline and 
within 1 week post intervention for biochemical analysis. High-density lipoprotein (HDL), 
low-density lipoprotein (LDL), triglycerides (TG), total cholesterol, fasting blood sugar and 
liver enzymes (alanine aminotransferase, ALT and aspartate aminotransferase, AST) are 
measured.  
 
Ultra-sonographic measurements including liver steatosis and shear wave elastograhy (SWE) 
are performed with the SuperSonic Imagine’s Aixplorer® Ultrasound machine (Super Sonic 
Image, Aix-en Provence, France). All measurements are performed by a single sonographer 
(Khairiah) where the inter-observer agreement level with another experienced sonographer 
(Juhara) is 85%. Hepatic steatosis is graded as normal (grade 0), mild (grade 1), moderate 
(grade 2) and severe (grade 3) based on visual analysis of intensity of echogenicity with an 
appropriate gain setting based on standardised paramete (13-14). Homogenous echo-
texture and echogenicity equal or slightly more than the renal cortex and spleen are 
characteristics of a normal liver. Liver echogenicity would be higher than the renal cortex 
and spleen if there was hepatic steatosis.  
 
Through the intercostal approach, SWE measurements are performed in the right liver lobe, 
at the supine position with the right arm in maximal abduction. The sonographer, assist by 
an ultrasonic time-motion image, located a liver portion of at least 6 cm thick, free of large 
vascular structures. Once the measurement area had been located, the sonographer presses 
the probe button to start an acquisition. Patients are asked to hold their breath for about 
five seconds, while the stiffness of the region of interest was measured and 10 
measurements are made for each patient and the median average value of those 
measurements is recorded in kilopascals (kPa: metric). Based on median liver elastography 
values, previously published four categories could be determined for different fibrosis stage. 
 
Safety consideration 
 
There may be risks to you if you participate in this study. These tests are fairly safe with litle 
risk involved. For most people, needle puncture for blood draws will cause discomfort but 
do not cause any serious problems. If any important new information is found during this 
study that may affect you wanting to continue to be part of this study, you will be told about 
it right away. 
 
Data management and statistical analysis 
 
Descriptive statistics are computed for all variables; means with standard deviations (SD) for 
continuous variables, median with interquartile range (IqR) for ordinal data and frequencies 
with percentages for categorical variables. Using Kolmogorov-Smirnov analysis, the data 
were normally distributed if the skewness is within the range of ±2 time standard error (SE) 

of skewness. Per-protocol (PP) and intention-to-treat (ITT) analysis are performed where 
comparison is made within-group (post- vs. pre-intervention for each intervention and 
control group) and between-group (MACR vs. control). Missing data are imputed using 
multiple imputation method. Independent t-test is used to analyse changes in weight, liver 
chemistry, lipid profile variables, fasting blood sugar and ultrasonographic changes of liver 
steatosis and fibrosis between the MACR and control group. SPSS version 22 (SPSS Inc., 
Chicago, United States) is used for all statistical analysis. All P values quoted were two-sided 
with P<0.05 considered as statistically significant. Sample size is 46 participants with 80% 
power of the study. 
 
Expected outcomes and benefit of the study 
 
This study is the first to determine that modified alternate day calorie restriction (MACR) is 
an effective dietary intervention compares to control group to help NAFLD individuals to 
lose weight, lower CAD risk and improving liver status. Specifically, we expected here that 
MACR regimen, which allowed participants to consume 30% of their energy needs on the 
fast day, resulted in a mean weight loss between 3-5% from baseline after 8 week of 
intervention thus it is also expected to decreases in several key biomarkers for CAD risk, 
such as total cholesterol, LDL cholesterol, triacylglycerols and blood pressure. Liver 
parameter which included liver enzymes, steatosis and fibrosis would also expect to 
improve after 8 weeks MACR. Additionally, we expected here that a similar rate of weight 
loss and adherence rate achieved during the MACR regime when compared with other 
alternate day calorie restriction regime. 
 
Problems Anticipated 
 
Compliance to dietary modification is one of the problems anticipated while conducting the 
trial. To overcome this problem, 2 weekly together with designated dietician given to 
participants in interventional group to monitor the calorie intake as well as education 
regarding calorie calculations. Besides that, we also will call participants once in a week to 
ensure the participants comply with dietary modification. 
 
Ethical Issues  
 
This study will be conducted with the highest respect for the subjects according to the study 
protocol, the ethical principles that have their origin in the Declaration of Helsinki, and the 
International Conference and Harmonisation (ICH) - Harmonised Tripartite Guideline for 
Good Clinical Practice (GCP). All researchers of this study will be GCP certified.  
 
The current clinical trial is approved by the Human Research and Ethics Committee of 
Universiti Sains Malaysia (reference: USM/JEPeM/15040117). 
 
 
1. Subject vulnerability  
 
1.1 The participants who involved in the study will be provided comprehensive explanation 
regarding the study procedure, any known adverse reactions, anticipated benefits and 
potential hazards of the study. If the subject is under my care as a doctor, the patient will be 
given full freedom to participate or not without affecting her medical condition 
management and care.  
 

1.2 Participants will not get vulnerable to the study as data obtained from this study does 
not identify participants individually. Besides that, the data will be independent and will not 
be used for any achievement assessment and decision related to work 
 
1.3 In addition, participants are given option to terminate their involvement in the study at 
any time without penalty or loss of benefits if they want to stop. A list of contact number of 
the investigators will be included in the participants information leaflet and they can directly 
contact the investigator if they have any queries or opt to cease the study.  
 
2. Declaration of absence of conflict of interest  
 
There is no conflict of interest presence in the study as all the participants are from 
Gastroentrology Clinic, Hospital USM. The researcher does not hold any supervisory or 
administrative post over the potential subjects that may influenced the participatory 
decision making.  
 
3. Privacy and confidentiality  
 
All participants’ data including informed consent, medical history, examination will be 
recorded on the documents prepared for the study. All forms are anonymous and will be 
entered into SPSS software. Data will be presented as grouped data and will not identify the 
responders individually. 
 
The medical information will be kept confidential by the study doctor and staff and will not 
be made publicly available unless disclosure is required by law. Data obtained from this 
study that does not identify participants individually will be published for knowledge 
purposes.  
 
4. Community sensitivities and benefits  
 
This study will benefit the participants as well as community. Participants may receive 
information about their liver status from examination to be done in this study and further 
treatment will be offer if indicated.  
 
The study allow us to explore the role of MACR in treating NAFLD in addition to exploring 
feature of NAFLD using new ultrasound techniques. Result and information from the study 
will provided evidences that modified alternate day calorie restriction (MACR) will be 
realible alternative ways to improve liver steatosis and fibrosis by weight loss compare to 
conventional ways. 
 
For community, the study allow us to later educate about NAFLD, including its risk, 
ultrasound diagnosis and role of modified alternate daily calorie restriction. Furthermore, 
the findings from study may allow us to apply for larger grants. 
 
5. Honorarium and incentives  
 
Honorarium including transportation cost will be given to the participants during the study.