Nitrogen balance (NB) is defined as the net difference between the intake of nitrogen (input) and its excretion (output). NB was considered as an indicator of the catabolic stress level. Thus, a negative NB can occur as a result of catabolism which leads to muscle mass loss. It was demonstrated that there is a relationship between meal frequency and N loss. Irregular meal patterns have been considered as a possible new risk factor for obesity and its consequences, specifically cardiovascular disease and metabolic syndromes. As the regular meal pattern is associated with higher BMI in many observational studies and has an impact on the metabolism of carbohydrates and lipids, it could also affect protein metabolism.

Eating irregularly can cause chrono-disruption because the changes in the timing of food intake as a consequence may also alter the chrono-biological or circadian rhythm of many hormones such as insulin, glucagon, adiponectin, leptin and gastric acid secretion. From this, investigators hypothesized that following an irregular meal pattern can negatively influence the N balance and circadian rhythm.

The study will have a crossover randomised, non blinded two meal pattern design with healthy adult female volunteers recruited from Nottingham, UK and the surrounding area.

Firstly, a screening visit lasting approximately an hour will be arranged and height, weight, and waist circumference will be measured following standard procedures (eg without shoes, wearing light clothes). Body composition will be obtained using skinfold thickness at four sites (triceps, biceps, subscapular and suprailiac). A blood sample (8 ml) will be obtained for routine blood investigation. A number of questionnaires will be completed to assess medical health, sleep duration, physical activity levels, eating behaviour and depression. On departure, subjects will be asked to complete a 4 days food diary.

The present study will last for 5 weeks. During the first week participants will be required to adopt one of two meal patterns, followed by a three week washout period, in which they will be allowed to return to their habitual diet, before undertaking a second week intervention following the second meal pattern. These meal patterns will consist of consuming 1) a regular meal pattern of 6 daily meals for two weeks, or 2) an irregular one consisting of consuming a different number of meals every day (between 3 and 9). Investigators propose to provide an iso-energetic diet containing identical foods with macronutrient composition (as a percentage of total energy per day) of 50% carbohydrate, 35% fat, and 15% protein for both patterns. All foods to be consumed during the study (1st and 5th week) will be provided free of charge. These will comprise foods commonly consumed in the British diet and will be consumed in amounts designed to keep body weight constant during the study. Participants will complete a food compliance diary over the study period. Physical activity will be captured by BodyMedia SenseWear armband to ensure that participants have a similar physical activity level in both intervention periods. This device records and analyses physiological parameters, and uses algorithms to report daily movement, calories burned, degree of physical activity and steps taken. It will be placed on the back of the upper left arm (the tricep). The Armband logo must face upward towards the shoulder and the silver sensors on the underside of the Armband will be in contact with skin.

Participants will be advised to consume a standardized evening meal based on their food record on the day before the start of each intervention and to provide 7, 24-h urine samples over the course of each intervention period. All urine was collected from study participants for 24 hr beginning at 7 am. Urine will be collected into 7 plastic urine containers without preservative. 5 ml will be stored from the well-mixed 24-h urine collection and analysed for urinary urea. In addition, they will be advised to wear a wireless iButton data logger to measure the wrist temperature in order to assess the circadian rhythm. The iButtons will be programmed to sample every 10 min, and will be attached to a double-sided cotton sport wrist band usingVelcro®, with the sensor face of the iButton being placed over the inside of the wrist, on the radial artery of the non-dominant hand.

Finally, subjective appetite (including hunger, fullness and desire to eat) will be assessed using visual analogue scales (VAS) during the intervention. Participants will be provided with a booklet in which to record the subjective appetite before and after each single meal on day 7 during both intervention periods when the subjects consume 6 meals/d during each intervention.

Inclusion Criteria:

• BMI between 18.5 and 25 kg/m2,
• Age between 18 and 40y,
• Non-smokers,
• Non high-alcohol consumers (≥ 14 units/week),
• Regular menstruation or on the oral contraceptive pills,
• Their weight is stable during the previous 3 months,
• No self-reported history of serious medical conditions and not under medication.

Exclusion Criteria:

• Pregnant or lactating women
• Smokers
• High-alcohol consumers (≥ 14 units/week)
• Subjects with high score for depression using Becks Depression Inventory
• subjects Eating Attitudes Test (EAT-26) score >20
• Subjects who on diet or seeking to lose weight
• Subjects with high consumption of coffee or tea > 3 cups/day

Only anonymised individual personal data will be shared, upon specific request from other researchers, for example, in order to undertake a meta analysis

when requested

On receipt of requests, data will be made accessible if agreed by the University of Nottingham