This randomised crossover study's primary aim is to investigate the effect of short-term fasting (eTRE) on subjective appetite and appetite-regulatory hormones (i.e., leptin, adiponectin, total glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), total peptide YY (PYY), acylated ghrelin and Insulin). In addition, to examine if the one-day early time-restricted eating influences energy expenditure and ad libitum energy intake in the periods following the standard meal test. The researchers will compare normal eating with early Time-Restricted Eating (eTRE) in healthy men.

Screening visit At the screening visit, the participant will firstly meet with a researcher involved in the study. They will then be directed to the laboratory before being asked to sign the informed consent form followed by the Health Screening questionnaire. Further, participants will have height and weight measured by a researcher, and BMI will be subsequently calculated. Those who are eligible to participate will be enrolled in the trial and will be invited for two experimental trials.

Those participants who are eligible will be asked to complete the International Physical Activity Questionnaire, to assess physical activity levels, a food preferences scale to examine how much they like the foods provided during experimental trials, and (should participants be eligible) participants will be provided with a food diary and scales that they must complete the day before their first and second experimental trials. Detailed instructions will be provided to participants on how to appropriately complete the food diary using the food scales.

Before experimental trials Participants will be instructed to complete two primary tasks prior to the experimental trials. First, participants will complete or follow a food diary. Specifically, 24 hours before the 2-day experimental trials, participants will complete a food diary, weighing and recording all food and beverage consumption. This is to ensure food and beverage intake is standardised in the 24 hours before the first experimental trial. Participants will also be instructed to fast from 20:30 the night before experimental trials.

Experimental trials Day 1 Participants will attend the lab at approximately 08:00 am. Participants will be fasting from 20:30 from the previous night (i.e., 12 hours without food or drink, except water). Once participants arrive, their weight will be recorded by a researcher, and they will rest until 09:00.

Normal diet In the normal diet experimental trial, the researcher will provide the standardised breakfast meal to participants at approximately 9:00 am. They will have 30 minutes to consume the meal. After this, participants will then rest in the metabolic research unit for the remaining 5.5 hours of the trial and will be allowed to work on laptops/tablets and relax in the metabolic research unit at the end of the trial (approximately 15:00; 7-hour trial in total), following a period of rest, they will then, at approximately 15:00, they will consume a standardised lunch, which they will have 30 minutes to consume. They will collect the standardised dinner to consume at home between 20:00 and 21.00 pm.

eTRE diet While in the eTRE diet experimental trial, they will consume the same meals but within a shorter time frame of six hours (9:00-15:00). The researcher will provide the standardised breakfast meal to participants at approximately 9:00 am. They will have 30 minutes to consume the meal. After this, participants will then rest in the metabolic research unit for the remaining 5.5 hours of the trial and will be allowed to work on laptops/tablets and relax in the metabolic research unit. Following a period of rest, they will then, at approximately 12:00 pm, they will consume a standardised lunch, which they will again have 30 minutes to consume. After this, participants will then rest in the metabolic research unit for the remaining 2.5 hours of the trial and will be allowed to work on laptops/tablets and relax in the metabolic research unit at the end of the trial (approximately 15:00; 7-hour trial in total), following a period of rest, they will then, at approximately 15:00 pm, they will consume a standardised dinner, which they will also have 30 minutes to consume. Water will be provided whenever the participant wishes. A researcher will be on hand to supervise the participant in the laboratory and will answer any questions the participant has.

The iVAS questionnaire will be administered to participants during the experimental trials (Normal/eTRE), which will take place seven times over a two-hour period. four of the questionnaires will be completed in the lab, while three will be taken home to fill out independently.

The design of the study is randomised and counter-balanced; half of the participants will complete the normal diet first, while the other half will complete the eTRE diet first.

Day 2 On the second day of the experimental trials, participants will arrive at the laboratory at approximately 08:00 having fasted (participants instructed not to eat or drink [except water] since 21:00 from the night of the normal diet experimental day and since 16:00 of the eTRE diet experimental day). A researcher trained in phlebotomy will insert a cannula into the antecubital vein. After about one hour to acclimatise to the cannula, a fasting blood sample will be collected, and data collection will begin.

Blood samples will be collected at various points. The investigators will first draw approximately 2 ml of excess blood and saline for each blood sample that may remain in the cannula. Following this, the investigators will withdraw 11.7 ml of blood for testing.

After the collection of each blood sample, measurements of the volume of oxygen consumed (V̇O2) and the volume of carbon dioxide produced (V̇CO2) will be recorded every 30 seconds for a total duration of 10-15 minutes. This will be achieved by the computerised open-circuit ventilated hood system (Quark RMR®, COSMED, Italy). Participants will be asked to lie supine and still. Once comfortable, a clear plastic canopy will be placed over the participant's head, and V̇O2 and V̇CO2 will be recorded every 30 seconds. Measurement values will be used to calculate the rate of fat and carbohydrate oxidation and the thermic effect of food through the tests. Volume and gas calibrations of the apparatus will be performed before measurement.

Participants will have a standardised breakfast (mixed meal tolerance test [1500 kcal]), consisting of croissants, butter, and milkshake.

Additionally, participants will be provided with an ad libitum buffet lunch in a private dining room under researcher supervision, as well as an ad libitum buffet dinner to take home and consume before going to bed. The lunch meal will include white and brown bread, butter, wafer chicken, mixed salad, yogurt, apples, bananas, muffins, and wine gums. The dinner meal will feature chicken, white and brown bread, cheese, butter, yogurt, nuts or granola, milk, chocolate chip cookies, apples and bananas. Participants will attend the laboratory for a supervised ad libitum buffet lunch. They will be seated individually and given a maximum of 30 minutes to consume the meal. All foods will be weighed before and after consumption to determine total energy intake. This lunch meal serves as the primary measure of energy intake in the study. More than enough items will be provided for the expected consumption, and if participants need more food items, they will be given them upon request. In addition, in the dinner meal, participants will be provided with ad libitum buffet takeaway dinner. They will be instructed to consume as much or as little of this meal as they wish, at any time before going to bed. It is important to inform them that they are not allowed to eat any of our foods for breakfast, the morning after Day 2. To determine the amount of each food item consumed, the investigators will measure the difference in weight between before and after the meal. The investigators will also use manufacturer details to determine energy and macronutrient consumption. Participants will return all remaining food packaging and containers the following day or by pre-arranged collection.

During this, appetite ratings (hunger, satisfaction, fullness, prospective food consumption and desire to eat) will be assessed using 100 mm Visual analogue scales (iVAS) at baseline, 0.5, 1, 2, 3, 4 and 5 hours. Additionally, a scale will be provided before and after the buffet dinner, resulting in a total of nine assessments. These scales are 100 mm long, with words anchored at each end expressing the most extreme appetite sensations.

Participants will then rest in the metabolic research unit, and they will be able to consume water ad libitum and will be able to bring laptops/tablets and books.

Finally, participants will be provided with the food diary they completed before the experimental trials (i.e., normal diet day and eTRE diet day) and the food scales.

In between 2-day experimental trials On the day before their experimental trials (i.e., normal diet day and eTRE diet day), participants will be asked to follow the food diary they completed 24 hours prior to each trial. They will also be instructed to fast from 8:30 PM the night before the experimental trials (i.e., no food or drink, except water, after 20:30). On the second day of the experimental trials. Participants should not eat or drink (except for water) after 9:00 pm the night of the normal diet experimental day and since 16:00 of the eTRE diet experimental day). Additionally, participants will be instructed to maintain their usual diet and daily routine and not to introduce any dietary changes or alterations to their routine that might affect appetite or appetite-related hormone responses. Participants will have the opportunity to contact the researcher at any point if they have questions.

Inclusion Criteria:

• Males aged 18 years or older.
• BMI between 18.5 and 24.9 kg/m²
• Have stable body mass for at least three months (within ±2 kg).

Exclusion Criteria:

• Females
• People who are younger than 18 or older than 65 years old.
• Following a special diet (e.g. weight loss, vegetarian or vegan, etc.).
• Have food allergies related to the study.
• Smoking.
• Taking any medications.
• Suffering from metabolic health issues, e.g., history of diabetes, cardiovascular disease, or eating disorders.

The stored data will be anonymous and follow the guidelines of the General Data Protection Regulation enforced in May 2018. Hard copies of participant information forms, including consent forms and dietary intake records, will be kept in locked filing cabinets. Keys to the filing cabinets will only be available to the primary investigator. All databases will be password protected and stored on a university computer with firewalls.

Participants' identifiable data will be stored in a locked cabinet. Digital data will be stored anonymously within the server of the University of Glasgow.

This project's Principal Investigator will be responsible for sharing the research data. Data will be preserved in Enlighten, the official online repository of Glasgow University's research output.

The data will be available when the study is completed. Data will be preserved for 10 years.

Data will be shared via the repository deposit (Enlighten). Only signatories of end-user agreements can access and use shared data.