The objective of this research is to determine if there is dose response of leucine on muscle maintenance during 4-week of alternate-day fasting (ADF) on blood parameters, body composition, mood and parameters related to appetite in young men with a body mass index of 23kg/m2 and above, whose body mass falls outside of the healthy range for their height (Asian reference point for increased risk of cardiovascular diseases). Participants have no diagnoses of, and are not prescribed medications for, cardiovascular, metabolic, diabetic, lung (except controlled asthma), kidney, or joint health conditions.

To achieve this objective, 100 hundred young men, aged between 21 and 35 years old will be recruited to complete a 4-week ADF programme. The fasting programme starts at 12 midnight and alternates between fasting and feeding days. Each fasting or feeding day is 24-hour in duration. During the fasting days, participants will abstain from all food and beverages except for ad libitum intake of plain water and other zero caloric beverages, and a small meal that is ~25% of daily caloric need. This small meal must be consumed during 12 pm to 2 pm in this study for standardisation to ensure consistency in total duration of fasting and feeding. During the feeding days, participants will eat up to ~125% of daily caloric need (calculated every week) with no restriction on the timing of meals as long as they are within the 24-hour period. The programme will end after 4 weeks (28 days) of continuous ADF.

All food for the 4-week ADF will be provided. Protein intake will be up to ~1.2 g/kg body mass/day on fasting day and up to ~2.4 g/kg body mass/day (~32-40% of calories) on feeding days. Participants will be randomly assigned (double blind) to the amount of leucine in their diet: 0.02, 0.04, 0.06 or 0.08 g/kg body mass per meal for 1 meal/day on fasting days and 3 meals/day on feeding days.

Blood parameters, body mass, height, waist-to-hip ratio, body composition, mood, stress level, and parameters related to eating and appetite will be measured before and after 4-week of ADF.

The investigators hypothesised a dose response in leucine to maintain muscle mass during weight loss.

Participation in this study requires a total of 4 weeks of ADF and 6 laboratory visits (sessions) spread over 5-6 weeks.

To participate in this study, participants will have to:

• complete the questionnaires regarding their health and diet and physical activity level (Global Physical Activity Questionnaire) on session 1,
• complete 5 weekly measurements of resting blood pressure and heart rate, fasting blood glucose (via finger prick blood sample), body mass, height, waist and hip circumferences, and body composition using bioelectrical impedance analysis (BIA) on sessions 1 and 3-6,
• complete the questionnaires regarding their mood [Brunel Mood Scale (BRUMS)], stress [Perceived Stress Scale (PSS)], food craving for general and specific foods [Food Craving Questionnaire-Trait Reduced (FCQ-T-r), and Food Craving Inventory (FCI)], eating behaviour [Three-Factor Eating Questionnaire-R18 - cognitive restraint component (TFEQ-r18)], and appetite, hunger, satiety and diet satisfaction (visual analogue scales, questionnaire and computerised test) twice on sessions 1 and 6,
• provide blood samples twice on sessions 1 and 6 for various biomarkers,
• complete 2 occasions of body composition measurement using dual-energy X-ray absorptiometry (DXA) on sessions 1 and 6,
• complete 4 weeks of ADF as instructed (details given below),
• consume only the food provided (high protein diet with leucine supplement) except for ad libitum intake of plain water and zero caloric beverages during the 4 weeks of ADF,
• complete daily log of food intake (including leftover/unconsumed food), physical activity and sleep during the entire 4 weeks of ADF,
• maintain a consistent diet between session 1 and the start of ADF,
• maintain a consistent physical activity level throughout the entire study. Do not start a new exercise programme during the entire duration of the study, and
• abstain from high level of physical activity throughout the entire study.

Prior to each session except session 2, participants are also required to:

• Abstain from any physical activity except activities of daily living for at least 48 hours.
• Abstain from alcohol and caffeine for at least 24 hours.
• Fast (i.e. do not eat or drink except plain water) overnight for 10 hours. In consideration of the fasting, these sessions will take place in the morning. To control for diurnal variation, the sessions will be timed such that the same measurements will be taken at approximately the same time of the day (+/- 1 hour difference).
• Obtain a full night of sleep the night before.

The above dietary and physical activity restriction is to minimise the confounding effects of diet and physical activities external to those conducted in this study.

Participants will also be instructed to:

• Inform the investigators immediately if they fall ill during the study or discover a health condition that requires them to consume any medication, steroids or hormones, and
• NOT to take calcium supplements or antacids (e.g. TUMS®) at least 24 hours prior to session 1, and NOT to schedule any scan that involves a contrast agent or ingestion of barium at least 14 days prior to sessions 1 and 6 to ensure accuracy of the DXA scans for body composition.

All sessions will be done in a fasted and rested state except session 2.

Session 1:

• Read and sign consent form

• Pre-screening:

  o Complete questionnaire on health and diet

  • Resting blood pressure and heart rate
  • Fasting blood glucose (via finger prick)
  • Body mass
  • Height

• Complete questionnaires on physical activity level, mood, stress, food craving, eating behaviour, appetite, hunger, satiety and diet satisfaction

• Antecubital blood sample

• Waist and hip circumference measurements

• Body composition using dual-energy X-ray absorptiometry and bioelectrical impedance analysis

Session 2: collection of 1 week of food. ADF will start within 10 days from session 1.

Session 3: 7 days (1 week) after the start of ADF

• Health measurements as in session 1:

  • Resting blood pressure and heart rate
  • Fasting blood glucose (via finger prick)
  • Body mass and height
  • Waist and hip circumferences
  • Body composition using bioelectrical impedance analysis

• Collection of 1 week of food

Session 4: 14 days (2 weeks) after start of fasting programme

• Same as session 3

Session 5: 21 days (3 weeks) after start of fasting programme

• Same as session 3

Session 6: 28 days (4 weeks) after start of fasting programme

• Same as session 1 except consent and pre-screening questionnaires on health and diet, and physical activity level are not applicable

Inclusion Criteria:

• Male
• 21 to 35 years old
• Do not smoke or use tobacco products (including shisha)
• Have a body mass index (BMI = body mass in kilograms divided by the square of height in metres) of 23 or greater

Exclusion Criteria:

• Unable to adhere to the requirements regarding physical activity and diet as stated in the research methodologies.
• Allergic or intolerant to milk and soy.
• Any other food allergies or dietary restrictions/preferences that the research team is unable to cater to due to constraints in food preparation and choices in order to control for composition of macronutrients, leucine content and calories in the food provided.
• Failed an exercise stress test.
• Have any health condition(s) that would be made worse by fasting.
• Taking long-term prescribed medication(s) for heart, blood (including blood sugar and blood pressure), lung (except controlled asthma), liver, kidney or joint condition(s).
• Taking other prescribed medication (including traditional Chinese medicine and other alternative medicine), drugs, steroids, or hormones that can affect any of the measurements in this study.
• Have electrical implant (such as pacemaker, implantable cardioverter defibrillator or neurostimulator), metal or plastic implant/rod/plate, or joint replacement in the body as these objects can affect the accuracy of the body composition measurement.

IPD sharing will be available upon request.

Individual participant data (IPD) will be made available to qualified researchers upon request. To access the data, researchers must submit a formal request, which will be reviewed and approved by the data sharing committee. Data access will be granted for research purposes only and will require adherence to data use agreements and ethics guidelines.