Subjects
This study was a randomized clinical trial, which was designed according to CONSORT guideline. Subjects were recruited from the Changsha area in China through advertisements in the vicinity of the Hunan Provincial People’s Hospital. A total of 285 adults with NAFLD that met the initial eligibility criteria were invited for a screening visit. NAFLD diagnosis was confirmed by abdominal ultrasound with liver stiffness > 9.6 kpa[18, 19]. Inclusion criteria were as follows: BMI >24 kg/m2; age between 18 and 65 years; stable body weight for 3 months prior to the beginning of the study (<5 kg weight loss or weight gain). Exclusion criteria were cardiovascular disease, uncontrolled hypertension, chronic inflammatory diseases, chronic infections, cancer, taking weight loss, lipid-, or glucose-lowering medications, and a history of bariatric surgery. Pregnant women and those planning a pregnancy or lactating were also excluded. All subjects provided written informed consent to participate in this study. The protocol was approved by the ethics committee of Hunan Provincial People’s Hospital (2018-37), and all research participants gave their written informed consent to participate in the trial.
Experimental Design
Block randomization was performed with a computer-generated random number sequence. An independent statistician generated the allocation sequence, and the study coordinator assigned the subjects to controlled dietary interventions trial for 12 weeks as the subjects enrolled. Subjects were randomised using the stratified random sample to groups: 1) control (n=79); 2) ADF (n=95); or 3) TRF (n=97) (Figure 1).
Dietary Intervention
During the dietary intervention period, individuals in the ADF group consumed 25% of their baseline energy needs through meals prepared in the metabolic kitchen of Hunan Provincial People’s Hospital, Changsha on the fast day (24 h), and then ate ad libitum at home on the feed day (24 h). Fast day meals were provided as a 3-day rotating menu, and were formulated based on the American Heart Association (AHA) guidelines (30% kcal from fat, 15% kcal from protein, 55% kcal from carbohydrate)[16, 20]. Energy needs for each subject were determined with the Mifflin equation. The feed and fast days began at midnight, and all fast day meals were consumed between 12.00 p.m. and 2.00 p.m. to ensure that all subjects were undergoing the same duration of fasting. Consumption of energy-free beverages, tea, coffee, and sugar-free gum was permitted and participants were encouraged to drink plenty of water. TRF subjects were provided with a meal within an 8-h window and asked to refrain from consumption of all food or beverages that included energy for the remaining 16 h. There were no additional instructions or recommendations on the amount or type of food consumed during the 8-h window. The timing of the feeding window during the day could be freely chosen to accommodate lifestyle habits of the participants. Subjects in the control group consumed 80% of their energy needs every day without any recommendations for or restrictions on their usual lifestyle patterns.
Outcome Measures
Anthropometric measurements were performed at the beginning of each week throughout the study period. Body weight was measured to the nearest 0.1 kg using a calibrated digital scale with the subjects wearing shoes and light clothing. Body height was determined to the nearest 0.1 cm using a wall-mounted stadiometer. Waist circumference was measured to the nearest 0.1 cm using a flexible tape placed midway between the lower costal margin and the super iliac crest during expiration. Body composition (fat mass and fat-free mass) was assessed by dual x-ray absorptiometry (DXA, Discovery-W version 12.6, Hologic, Bedford, MA, USA).
The value of liver stiffness was measured by Fibrocan (Echosens Corp., Paris, France). A total of 10 measurements were carried out, and liver stiffness value was record only if the interquartile range did not exceed 40% in any of the measurements. The results were expressed in kilopascals. The median value was taken as representative[18, 19].
Fasting blood samples were collected at 6.00 a.m. at baseline and at week 4 and week 12 after initiation of the intervention. Participants were instructed to avoid exercise, alcohol, and coffee for 24 h before each visit. The collected blood was centrifuged for 10 min at 520 × g at 4°C to separate plasma from red blood cells and was stored at -80°C until analysis. Plasma total cholesterol, direct LDL cholesterol, HDL cholesterol, and triglyceride concentrations were measured in duplicate with enzymatic kits (Biovision, Mountainview, CA, USA). LDL particle size was determined by linear polyacrylamide gel electrophoresis (Quantimetrix Lipoprint System, Redondo Beach, CA, USA).
A validated visual analog scale (VAS) was used to measure hunger, fullness, and satisfaction with the study diet[21]. The scale was completed at baseline and at week 4 and week 12. The VAS consisted of 100-mm lines and subjects were asked to place a vertical mark across the line corresponding best to their feelings of hunger, satisfaction, and fullness, with the scale ranging from 0 (not at all) to 100 (extremely). Quantification was performed by measuring the distance between the left end of the line and the vertical mark.
Statistical Analysis
All statistical analyses were performed using the Statistical Package for the Social Sciences software (SPSS version 19.0, IBM, Armonk, NY, USA). Numerical data were reported as mean ± standard deviation and/or range. Continuous variables were compared using one-way analysis of variance (ANOVA) and categorical variables using Fisher’s exact or Pearson chi-square tests. A P-value < 0.05 was considered statistically significant.