Dose Response of Leucine on Muscle Maintenance During Weight Loss

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.

Study Overview

• Status: Completed
• Conditions: Overweight and Obese Adults; BMI >= 23
• Intervention / Treatment: Dietary supplement: leucine; Dietary supplement: leucine; Dietary supplement: leucine; Dietary supplement: leucine

Detailed Description

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

• Study Type: Interventional
• Enrollment (Actual): 92
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Singapore
  • Singapore, Singapore, 637616
    • Nanyang Technological University

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: No

Description

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.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Treatment
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Triple

Number of Arms

4

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: 0.02g/kg body mass per meal/day; In this study, participants would be randomly (by chance) assigned 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.	Dietary supplement: leucine; 0.02g/kg body mass/meal/day; Dietary supplement: leucine; 0.04g/kg body mass/meal/day; Dietary supplement: leucine; 0.06g/kg body mass/meal/day; Dietary supplement: leucine; 0.08g/kg body mass/meal/day
Experimental: 0.04g/kg body mass per meal/day; In this study, participants would be randomly (by chance) assigned 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.	Dietary supplement: leucine; 0.02g/kg body mass/meal/day; Dietary supplement: leucine; 0.04g/kg body mass/meal/day; Dietary supplement: leucine; 0.06g/kg body mass/meal/day; Dietary supplement: leucine; 0.08g/kg body mass/meal/day
Experimental: 0.06g/kg body mass per meal/day; In this study, participants would be randomly (by chance) assigned 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.	Dietary supplement: leucine; 0.02g/kg body mass/meal/day; Dietary supplement: leucine; 0.04g/kg body mass/meal/day; Dietary supplement: leucine; 0.06g/kg body mass/meal/day; Dietary supplement: leucine; 0.08g/kg body mass/meal/day
Experimental: 0.08g/kg body mass per meal/day; In this study, participants would be randomly (by chance) assigned 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.	Dietary supplement: leucine; 0.02g/kg body mass/meal/day; Dietary supplement: leucine; 0.04g/kg body mass/meal/day; Dietary supplement: leucine; 0.06g/kg body mass/meal/day; Dietary supplement: leucine; 0.08g/kg body mass/meal/day

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Body mass	Body mass measured using BIA weighing scale	Start and end of 4-week intervention and weekly during intervention
Lean mass	Lean mass measured using DXA	Start and end of 4-week intervention
Appendicular Lean Mass	Sum of lean mass in upper and lower limbs measured using DXA	Start and end of 4-week intervention
Body mass index	body mass in kilograms divided by sqaure of height in metres	Start and end of 4-week intervention and weekly during intervention
Lean mass index	lean mass in kilograms divided by square of height in metres	Start and end of 4-week intervention
Appendicular lean mass index	Appendicular lean mass in kilograms divided by square of height in metres	Start and end of 4-week intervention
Fat mass	Fat mass	Start and end of 4-week intervention and weekly during intervention
Percentage body fat	Percentage body fat	Start and end of 4-week intervention and weekly during intervention
Fat mass index	Fat mass in kilograms divided by square of height in metres	Start and end of 4-week intervention and weekly during intervention
Trunk fat	Fat mass around the trunk	Start and end of 4-week intervention
Appendicular fat mass	Sum of fat mass in the upper and lower limbs	Start and end of 4-week intervention
Appendicular fat-free mass index	Appendicular fat-free mass in kilograms divided by square of height in metres	Start and end of 4-week intervention and weekly during intervention
Resting systolic blood pressure		Start and end of 4-week intervention and weekly during intervention
Resting diastolic blood pressure		Start and end of 4-week intervention and weekly during intervention
Fasting blood glucose		Start and end of 4-week intervention and weekly during intervention
HbA1c		Start and end of 4-week intervention
Insulin		Start and end of 4-week intervention
The HOMA-IR index	Homeostatic Model Assessment of Insulin Resistance index	Start and end of 4-week intervention
Total cholesterol		Start and end of 4-week intervention
HDL cholesterol		Start and end of 4-week intervention
LDL cholesterol		Start and end of 4-week intervention
Total cholesterol to HDL cholesterol ratio		Start and end of 4-week intervention
Triglycerides		Start and end of 4-week intervention

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Android fat mass		Start and end of 4-week intervention
Gynoid fat mass		Start and end of 4-week intervention
Bone mineral content	Bone mass	Start and end of 4-week intervention
Bone mineral density	Bone mineral density	Start and end of 4-week intervention
Waist Circumference	Waist Circumference	Start and end of 4-week intervention and weekly during intervention
Hip circumference		Start and end of 4-week intervention and weekly during intervention
Waist-to-hip ratio	Waist circumference to hip circumference ratio	Start and end of 4-week intervention and weekly during intervention
Fat free mass	Fat free mass measured using BIA	Start and end of 4-week intervention and weekly during intervention
Skeletal muscle mass	Skeletal muscle mass measured using BIA	Start and end of 4-week intervention and weekly during intervention
Appendicular fat-free mass	Sum of fat free mass in upper and lower limbs measured using BIA	Start and end of 4-week intervention and weekly during intervention
Visceral fat area	Visceral fat area measured using BIA	Start and end of 4-week intervention and weekly during intervention
Fat-free mass index	Fat-free mass in kilograms divided by square of height in metres	Start and end of 4-week intervention and weekly during intervention
Skeletal muscle mass index	Skeletal muscle mass in kilograms divided by square of height in metres	Start and end of 4-week intervention and weekly during intervention
Mood	Brunel Mood Scale (BRUMS). BRUMS is the modified and shorter version of the Profile of Mood Stats (POMS). The 24-item BRUMS was used to assess mood responses. It has six subscales (tension, depression, anger, vigour, fatigue, and confusion) of four mood descriptors each, with respondents rating how they feel "right now" on a 5-point Likert-type scale (0 = not at all, 1 = a little, 2 = moderately, 3 = quite a bit; 4 = extremely). During its development, the BRUMS showed robust psychometric properties across samples of adult students, adult athletes, young athletes, and schoolchildren, and has since demonstrated psychometric integrity for use with many other population groups (doi.org/10.3390/su13116116).For each subscale, the raw score are summed and converted to a T-score based on the norms published in the paper (doi.org/10.3390/su13116116). Higher T-score for each subscale reflects higher profile of that subscale (i.e. tension, depression, anger, vigour, fatigue, or confusion).	Start and end of 4-week intervention
Stress level	Perceived Stress Scale (PSS). The 10-item PSS asks participants about feelings and thoughts during the last month, with responses ranging from 0 (never) to 4 (very often). The final score obtained by reversing responses (e.g., 0 = 4, 1 = 3, 2 = 2, 3 = 1 & 4 = 0) to the four positively stated items (items 4, 5, 7, & 8) and then summing across all scale items. Higher scores indicating higher levels of perceived stress.	Start and end of 4-week intervention
Food craving - habitual	The Food Craving Questionnaire-Trait Reduced (FCQ-T-r) measures the habitual (unspecified time frame) trait craving for food in general. It predicts food cravings in daily life. It represents a stable trait but is also sensitive to changes in eating behaviour. The questionnaire consists of 15 questions, participants are required to rate the frequency of each statement with a 6-likert scale (never or not applicable = 1, to always = 6). By summing up the scores of the scales, higher scores represent higher level of food cravings.	Start and end of 4-week intervention
Food craving - specific foods	Food Craving Inventory (FCI) measures food craving for specific foods and the versions validated for use in United States and United Kingdom were used. Questionnaire items pertaining to specific local foods were also added for a combined total of 61 food items (i.e. questionnaire items). For each item, participants were asked how often they have experienced a craving for that particular food on a 5-point Likert scale (never = 1, to always/almost every day = 5). The higher the score, the higher the food craving.	Start and end of 4-week intervention
Eating Behaviour	Three-Factor Eating Questionnaire-R18 - cognitive restraint component (TFEQ-r18 Cog). It has 6 items and each scored 1 to 4 for a total of 24 points. The higher the total score, the more disordered the eating behaviour.	Start and end of 4-week intervention
Baseline appetite	Baseline appetite using visual analogue scale 0 to 100. The higher the score, the higher the baseline appetite	Start and end of 4-week intervention upon arrival in the laboratory
Hunger over past 4 weeks	Hunger over past 4 weeks. Visual analogue scale 0 to 100. The higher the number the greater the amount of hunger.	Start (i.e. over the past 4 weeks before start of the intervention) and end of 4-week intervention (i.e. over the past 4 weeks during the intervention)
Satiety over past 4 weeks	Satiety over past 4 weeks. Visual analogue scale 0 to 100. The higher the number, the greater amount of satiety.	Start (i.e. over the past 4 weeks before start of the intervention) and end of 4-week intervention (i.e. over the past 4 weeks during the intervention)
Diet satisfaction over past 4 weeks	Diet satisfaction over past 4 weeks. Visual analogue scale 0 to 100. The higher the number the greater the satisfaction.	Start (i.e. over the past 4 weeks before start of the intervention) and end of 4-week intervention (i.e. over the past 4 weeks during the intervention)
Implicit satiety goal	Implicit satiety goal (unconscious, habitual, or subjective levels of fullness that individuals aim to achieve when eating) using visual analogue scale 0 to 100. The higher the number, the greater implicit satiety goal.	Start and end of 4-week intervention
Ideal portion size	Ideal portion size (IPS). Participants were presented with a diverse range of 25 foods typically consumed in Singapore. The computerised portion selection tasks presented a series of 50 images of each food that increased in portion size in 20 kcal increments (20-1000 kcal). Participants could increase or decrease the portion size by 20 kcal using the left and right arrow-keys on the keyboard and were encouraged to view the full range of portions before selecting their ideal portion. Foods were presented in a randomised order and an average portion size for each participant was calculated by averaging the selected portion sizes of all 25 foods (α = 0.95). This created an overall index of IPS.	Start and end of 4-week intervention
Expected satiety	Expected satiety (ES) measured using computerised task. The ES task followed the same format as the IPS task, in other words, 50 images were presented depicting 20 kcal increment changes in portion size. This task sought to measure participants' expectations about how satiating a diverse range of foods were. Larger portions selected indicated that participants require larger quantities of the food to delay the onset of hunger, in other words they have lower expected satiety. The same 25 foods presented in the IPS were also presented in the ES in a randomised order and were averaged to create an overall index of ES (α = 0.95).	Start and end of 4-week intervention
Food-related characteristics	A single image of each food item was presented to participants alongside questions of food-related characteristics. Participants were asked to rate how filling they expected each food to be (not filling at all (0) to very filling (100)), how much they like each food in general (not at all (0) to very much (100)) and how often they eat each food (never (0) to more than once a week (5)). Each food was presented in a randomised order and average scores across all 25 foods for participants perceptions of how filling, frequently consumed and preference for foods were calculated.	Start and end of 4-week intervention

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Height	Height - used to calculate body mass index and other indices	Start and end of 4-week intervention and weekly
Hydration status	Intracellular water (litre)/extracellular water (litre). This ratio is a reflection of the hydration status in the body. Hydration status can affect the body composition measurements using BIA.	Start and end of 4-week intervention and weekly during intervention
SGP/ALT	blood biomarker of liver function	Start and end of 4-week intervention
SGOT/AST	blood biomarker of liver function	Start and end of 4-week intervention
Serum sodium		Start and end of 4-week intervention
Serum potassium		Start and end of 4-week intervention
Serum urea		Start and end of 4-week intervention
Serum creatinine		Start and end of 4-week intervention
Insulin growth factor-1		Start and end of 4-week intervention
Full blood count		Start and end of 4-week intervention
Weekly physical activity level	Participants complete the type, intensity and duration of each physical activity during the week similar to Global Physical Activity Questionnaire (GPAQ) developed by the WHO. Each activity is calculated in Metabolic Equivalent (MET) value. Moderate MET value is 4.0 and vigorous MET value is 8.0 while for transport domains MET value is 4.0.	1-2 weeks before the start of the intervention and for each week of the 4-week intervention
Food diary	Participants have to record daily whether they finish the food provided, including the portion of leftovers if any. On fasting days, they also log the timing of their small meal	Daily during the 4-week intervention
Sleep diary	Participants have to log their sleep time, wake up time and subjective sleep quality daily	Daily during the 4-week intervention

Collaborators and Investigators

• Sponsor: Nanyang Technological University
• Collaborators: Ministry of Education, Singapore

Study record dates

Study Major Dates

• Study Start (Actual): August 18, 2020
• Primary Completion (Actual): February 11, 2021
• Study Completion (Actual): February 11, 2021

Study Registration Dates

• First Submitted: April 26, 2026
• First Submitted That Met QC Criteria: May 6, 2026
• First Posted (Actual): May 13, 2026

Study Record Updates

• Last Update Posted (Actual): May 13, 2026
• Last Update Submitted That Met QC Criteria: May 6, 2026
• Last Verified: April 1, 2026

More Information

Terms related to this study

• Keywords: Obesity; Overweight; appetite; Protein; mood; Skeletal muscle; Intermittent fasting; Leucine; food craving; dose response; Alternate Day Fasting; ADF; quality weight loss; muscle maintenance; blood health markers
• Additional Relevant MeSH Terms: Nutrition Disorders; Overnutrition; Body Weight; Pathological Conditions, Signs and Symptoms; Behavior; Nutritional and Metabolic Diseases; Signs and Symptoms; Feeding Behavior; Fasting; Overweight; Obesity; Intermittent Fasting; Amino Acids, Peptides, and Proteins; Amino Acids; Amino Acids, Essential; Amino Acids, Branched-Chain; Leucine
• Other Study ID Numbers: IRB 2019-07-039

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: IPD sharing will be available upon request.
• IPD Sharing Access Criteria: 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.
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; SAP; ICF

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No