Time Restricted Eating and Meal Frequency for Appetite Control

Number of Participants:

10 adults (males and females) who have expressed an interest in taking part and meet the inclusion criteria. Should there be any withdrawals, the investigators will continue to replace participants until 10 have completed the trial.

The primary objective of this study was to explore the efficacy and physiological responses to a controlled time-restricted eating regime and dietary interventions (2 or 5 meals per day) on the participant's appetite response, measured with visual analogue scales (VAS). VAS is completed on test days (recorded at time points 0, 30, 60, 90, 120, 150 and 180 minutes) and on all other study days (recorded hourly during waking hours). The secondary objective is to compare the influence of dietary interventions (2 or 5 meals per day) on the following: plasma fasting blood glucose and 3 hours post-prandially after a standardized test meal. Analysed by KONE for timepoints 0 and 180 mins. Plasma fasting insulin and 3 hours post-prandially after a standardized test meal. Analysed by ELISA for timepoints 0 and 180 mins. Continuous glucose monitoring System (CGMS) on all study days to assess free-living glycaemic control in response to diet. All components of energy expenditure and substrate utilization are monitored by ventilated hood (indirect calorimetry) as resting metabolic rate (RMR, 30 minutes before eating breakfast); thermic effect of food (TEF postprandial 3 hours, measured 10 minutes every 30 minutes by the ventilated hood). Moreover, to assess changes in physical activity, with accelerometry on all study days and the Baecke Physical Activity Questionnaire (BPAQ) completed on each test day. To assess if meal frequency influences sleep, a sleep diary will be completed on all study days and the Epworth Sleepiness questionnaire (ESQ) completed on each test day. Finally, to assess if meal frequency influences body mass and body composition (fasted body weight measured on all food collection and test days), and fasted waist and hip circumferences and fat mass by bio-impedance analysis (BIA) measured on all test days.

A randomized crossover trial (RCT). Each participant will complete a 21 day study design as follows:

• 7 days food diary (recording at home, habitual diet) [Days -6 to 0].
• 2 days maintenance diet 1 (MT1) [Days 1-2].
• Test Day on Day 3.
• 4 days intervention weight loss diet (2MPD, 2 meals/day or 5MPD, 5 meals/day) [Days 3-6].
• Test Day on Day 7.
• 8 days washout period, no meals provided [Days 7-14].
• 2 days maintenance diet 2 (MT2) [Days 15-16].
• Test Day on Day 17.
• 4 days intervention weight loss diet (5MPD, 5 meals/day or 2MPD, 2 meals/day) [Days 17-20].
• Test Day on Day 21.

Study Diets:

During the screening visit, RMR was measured for each participant, and based on the results, each participant received a different meal portion. After receiving the RMR results for each participant, the kitchen staff at the Human Intervention Studies Unit (HISU) were emailed the participant number, RMR results, and the randomised diet they would start (2MPD or 5MPD), allowing the dietitian to assign the closest diet on the diet scales (1250, 1500, 1750, 2000, 2250 kcal). For example, if the RMR result for a volunteer were 1800 kcal, it would be put in the closest diet, 1750 kcal.

All food will be prepared at the Human Intervention Studies Unit (HISU), Rowett Institute.

This study will include two types of diet:

• A maintenance diet (MT, 15:30:55% of energy from protein, fat and carbohydrate respectively). On days 1-2 (MT1) and 15-16 (MT2) this diet will be fed to meet measured energy requirements (1.5 x Resting Metabolic Rate, RMR).
• A high protein (HP) intervention weight loss diet (WL, 30:35:35% of energy from protein, fat and carbohydrate respectively). On days 3-6 (WL1) and 17-20 (WL2) this will be fed to 100% resting metabolic rate. The food intake during the intervention periods will be randomized for two patterns fed between 07:00 to 17:00; five small meals per day (5MPD) and two large meals per day (2MPD). The protein in the diets will come from different sources like meat, poultry, fish, soy, and beans. In addition, all meals on the diets will contain equal proportions of protein so that the daily protein is not all consumed in one meal.

Study Measurements:

In total there will be nine study visits to the Human Intervention Studies Unit (HISU), Rowett Institute: one screening visit, which last approximately 2 hours, 4 visits for Test Days representing pre and post dietary phases (day 3rd, 7th, 17th, and 21st), which last approximately 4-5 hours, and 6 visits to collect food (day 1st, 3rd, 6th, 15th, 17th, and 20th), two of them are at the same day of Test Day, which last approximately 30 mins, except 2 visits will be 4-5 hours because it is the day of Test Days too. All visits details are described in the protocol. Researcher(s) carrying out the procedure: The study will be co-ordinated by a Research Team, consisting of Professor Alexandra Johnstone (PI), Prof Justin Rochford (CI), Ms Nouf Alkhattabi (PhD student), Claire Fyfe (Research Technician), David Bremner (Research Assistant), Claire Kidd (Research Assistant), Christina Petkov (Honorary Research Nurse).

Screening Visit:

The participant should already have a copy of the PIS in advance of the consent and screening visit should already have a good understanding of the study and have sufficient time to read it before this visit. After discussion, the consent should always be obtained before the health status and eligibility. n=10 to complete.

All measurements will be conducted after a fast from 10:00 pm the night before. Breakfast will be provided at the end of the screening visit.

Screening will continue until study completion and involves.

• Completing written, informed consent paperwork (PIS and consent form).
• A self-report general health screening questionnaire.
• RMR measurement for calculation of energy requirements (ventilated hood).
• Height and weight measurements to calculate body mass index (BMI).

The participants will then be given a 7-day weighed intake food diary and scales to take away with them to complete to establish their regular eating habits before the start of diet provision.

Test Day Visits (on Days 3, 7, 17 and 21):

All measurements will be conducted after a fast from 10pm the night before. On days 3 and 17 meals will be provided as breakfast, lunch, dinner, and snacks according to the intervention diet randomisation (2MPD or 5MPD), while on days 7 and 21 only breakfast will be provided.

• Overnight fasted anthropometric measures
• Body weight and body mass index (BMI) calculation.
• Total body fat (TBF), fat mass (FM) and fat-free mass (FFM) by bio-impedance analysis (BIA).
• Waist and hip circumferences measured using a tape measure.
• Resting energy expenditure (RMR) will be measured for 30mins in fasting state by indirect calorimetry.
• The blood samples will be collected by multiple venepuncture (T0 and T180mins).
• Venepuncture for fasted blood sampling (one 4ml LiHep tube):
• T0mins Glucose (1x500μl plasma in KONE cup).
• T0mins Insulin (1x300μl plasma in 0.5ml Eppendorf).
• T0mins LiHep Spare (2x300μl plasma in 1.5ml Eppendorf).

The total volume of blood taken will be as follows:

• In each visit, 8ml will be taken for each participant.
• The total blood taken for 4 visits will be 32ml for each participant.
• Measure of fasted participant appetite by Visual Analogue Scales (VAS) at T0mins.
• Consumption of intervention breakfast meal at T0.
• After the test meal the thermic effect of food (TEF) will be calculated from indirect calorimetry measurements taken for the last 10 min of every 30 min up to 3 hours (T20, T50, T80, T110, T140 and T170mins).
• Questionnaires:
• A validated self-report questionnaire for physical activity Baecke Physical Activity Questionnaire (BPAQ) and the sedentary is categorized as, low, medium, or high.
• Assessment of how meal frequency influences sleep using the Epworth Sleepiness questionnaire (ESQ).
• Post-prandial measurement of participant appetite by VAS at T30, T60, T90, T120, T150 and T180mins.
• Venepuncture for postprandial blood sampling (one 4ml LiHep tube):
• T180mins Glucose (1x500μl plasma in 0.5ml KONE cup).
• T180mins Insulin (1x300μl plasma in 0.5ml Eppendorf).
• T180mins LiHep Spare (2x300μl plasma in 1.5ml Eppendorf).
• Food diary recorded for the remainder of the day.

Other Measurements:

• Continuous Glucose Monitoring System (CGMS) throughout the 3week study (two, 2week insertions per participant) using Freestyle Libre devices. Sensors record data for 14 days at a time. Freestyle Libre sensor: water resistant sensor (in up to 1m water for a maximum of 30mins) that continuously measures the glucose concentration in interstitial fluid, and stores 8 hours of data. Can be scanned by any mobile device capable of downloading the FreeStyle LibreLinkUp app. LibreLinkUp is compatible with Android and iOS smartphones which meet the following requirements: Android OS 6.0 (or higher) or iOS 11 (or higher) and an internet connection. Participants without a smartphone capable of using the app will be provided with a budget smartphone. Each sensor has a working life of 2 weeks. Participants will be supplied with the required number of sensors in order to complete the study. The FreeStyle Libre sensor is applied on to the back of the upper arm with a simple, disposable device called an applicator. When the sensor is applied, a thin, flexible and sterile fibre is inserted just under the skin. It is held in place with a small adhesive pad. In a study conducted by Abbott, users agreed that inserting the sensor was no more painful than a typical finger prick test. To obtain a glucose reading from this device, participants must perform a quick, painless 1-second scan of the reader over the sensor. This scan gives the investigators more information than monitoring with blood glucose test strips, without the need for routine finger pricks. Each scan of the reader over the sensor gives a current glucose reading, the last 8-hours of glucose history, and a trend arrow showing if glucose is going up, down, or changing slowly. The reader can scan through clothing.
• Temporal changes in appetite using VAS questionnaires throughout the 2 intervention phases of the study. Questionnaires completed hourly during waking hours.
• Composition of all meals consumed during the 3weeks study. Recorded by participants using weighback sheets and the food scales provided at the screening visit.
• Physical activity levels recorded by Actigraph accelerometers. The actigraph provides data for energy expenditure per day, the intensity of the activity carried out taken when the monitor was worn.
• Assessment of how meal frequency influences sleep. Hours slept will be calculated from the data recorded by the Actigraph accelerometers and from two, 7day sleep diaries with questions the participants will answer every morning of the intervention phases.