The objectives of this study are to examine the effects of protein supplementation on 4 weeks of alternate-day fasting (ADF) on fasting blood glucose, resting blood pressure, and body composition in young Asian men with unhealthy body mass index ≥ 23 kg/m2.

To achieve the objective, 40 young men aged 21 to 35 years old will be recruited to participate in this study and 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. On fasting days, no food and beverages are allowed except for ad libitum intake of plain water and/or other zero-calorie beverages, and a small meal between 400-600 kcal. In this study, participants would be randomly assigned to either the control or protein supplementation group (20 in each group). The control group will consume a small meal of their choice as long as it is between 400-600 kcal while the protein supplementation group will consume a small meal between 400-600 kcal that will include a protein supplement that would be provided (125 kcal, 25 g protein) and with the remainder of the meal of their choice. For both groups in this study, the small meal is to be consumed during 12 pm to 2 pm for standardisation to ensure consistency in total duration of fasting and feeding. The programme will end after 4 weeks (28 days) of continuous alternate-day fasting.

Fasting blood glucose, resting blood pressure and heart rate, body mass, height, and body composition will be measured before and after 4-week of ADF. In addition, these measurements, except body composition, will be repeated at the end of each week of ADF.

The research hypothesis are: 1) 4 weeks of ADF will reduce body mass via both fat and muscle loss, and improve health indices as measured by fasting blood glucose and resting blood pressure in young individuals with BMI greater than the desired healthy range, and 2) protein supplementation during fasting days will reduce muscle loss during loss of body mass.

Methods Overview Participation in this study requires a total of 4 weeks of alternate-day fasting and 5 laboratory visits (sessions) spread over 5 weeks.

To participate in this study, participants will have to 1) sign the consent form, 2) complete a questionnaire which assesses their health with respect to the inclusion and exclusion criteria to ensure their safety and participation eligibility, 3) complete weekly questionnaire about their physical activity level, 4) complete 5 times of a health screening (measurement of resting blood pressure and heart rate, fasting blood sugar, body mass and height), 5) complete 2 occasions of body composition measurement, 6) complete 4 weeks of alternate-day fasting programme, and 7) complete 4 x 2-day dietary recalls for a total of 8 days of dietary recalls. Participants will also be required to consume a protein supplement on fasting days if assigned to the protein supplementation group.

Participants are required to:

1. follow the fasting programme as instructed including the protein supplementation if assigned (details given below),
2. abstain from taking medication (including traditional Chinese medicine) not previously declared unless when ill (participants are instructed to inform the investigators immediately if they fall ill during the study), hormones, steroids, and any supplements external to the study, including protein, vitamin and mineral supplements during the entire duration of the study,
3. maintain a consistent physical activity level throughout the entire duration of the study,
4. abstain from any physical activity except for activities of daily living for at least 48 hours prior to each session,
5. abstain from alcohol and caffeine for at least 24 hours prior to each session,
6. fast overnight for 10 hours before each session. In consideration of the fasting and to control for diurnal variation, all sessions will take place in the morning, and
7. abstain from all food and beverages during each session except for plain water that you may drink as much as you need.

Session 1: Consent, Questionnaires, Health Screening and Body Composition

Procedures involved:

1. Read and sign consent form if participating
2. Complete Subject Screening Questionnaire (to assess participants' health with respect to the inclusion and exclusion criteria to ensure their safety and participation eligibility)
3. Complete Global Physical Activity Questionnaire (GPAQ; to assess participants' physical activity level)
4. Answer questions related to health with respect to the inclusion and exclusion criteria
5. Have resting blood pressure and heart rate, body mass, and height measured
6. Have fasting blood sugar level measured using finger prick sampling
7. Undergo a direct segmental multi-frequency bioelectrical impedance analysis (DSM-BIA) to determine body composition
8. Collect the weekly supply of protein supplement in the form of a protein powder to be mixed with a beverage of their choice and to be consumed during the fasting days (see below) for participants assigned to the protein supplementation group.

Alternate-Day Fasting for 4 weeks (start within 1 week from session 1)

Procedures involved:

1. Programme starts at 12 midnight
2. Alternate between fasting and feeding days
3. Each fasting or feeding day is 24-hour in duration
4. On fasting days: no food and beverages except for ad libitum intake of plain water and/or other zero-calorie beverages, and a small meal between 400-600 kcal. In this study, participants would be randomly (by chance) assigned to either the standard or protein supplementation group. The standard group will consume a small meal of their choice as long as it is between 400-600 kcal while the protein supplementation group will consume a small meal between 400-600 kcal including the protein supplement (125 kcal, 25 g protein) that is provided and with the remainder of the meal of their choice.
5. For both groups in this study, this small meal is to be consumed during 12 pm to 2 pm for standardisation to ensure consistency in total duration of fasting and feeding. One must finish the protein supplement during this period of time if assigned to the protein supplementation group.
6. On feeding days: ad libitum eating except no alcohol and caffeine for at least 24 hours prior to sessions 2 to 4 if coinciding with the feeding days.
7. Programme ends after 4 weeks (28 days) of continuous alternate-day fasting

Sessions 2 to 4: Dietary Recalls, Health Screening and Questionnaire (at the end of each week of alternate-day fasting, ~1 hour for each session)

Procedures involved:

1. Complete a 2-day dietary recall on any 2 fasting days for the past week prior to each session
2. Have resting blood pressure and heart rate, body mass, and height measured
3. Have fasting blood sugar level measured using finger prick sampling
4. Complete Global Physical Activity Questionnaire (GPAQ)
5. Collect weekly supply of protein supplement if assigned to the protein supplementation group

Session 5: Dietary Recalls, Health Screening, Questionnaire and Body Composition (last session after 4 weeks of alternate-day fasting, ~1.5 hours)

Procedures involved:

1. Complete a 2-day dietary recall on any 2 fasting days for the past week prior to the session
2. Have resting blood pressure and heart rate, body mass, and height measured
3. Have fasting blood sugar level measured using finger prick sampling
4. Complete Global Physical Activity Questionnaire (GPAQ)
5. Undergo a direct segmental multi-frequency bioelectrical impedance analysis (DSM-BIA) to determine body composition

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:

One cannot participate in or will be released from this study if any of the following conditions or situations applies to them:

• Unable to adhere to requirements regarding physical activity and diet as stated in the methods
• 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 glucose and blood pressure), lung (except controlled asthma), liver, kidney or joint condition(s)
• Taking other long-term prescribed medication (including traditional Chinese Medicine and other alternative medicine) that are known to affect any of the measurements in this study
• Allergic or intolerant to milk and soy (the protein supplement that participant may be required to consume, contains milk proteins and traces of soy lecithin and lactose)
• Have an artificial electrical implant such as a defibrillator or pacemaker

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.