Time-restricted Feeding Improves Basal Fat Oxidation and Body Composition But Not Fat Oxidation During Exercise

Primary outcomes

1. Measurement of body composition (in kg) with Dual-energy X-ray absorptiometry (DXA)

   Time frame: 9 months

   All participants underwent a whole-body composition scan with light clothing to measure total body mass (kg), fat mass (kg), fat-free mass (kg), and lean body mass (kg), with an automatically chosen scanning mode by the DXA machine (Lunar Prodigy Pro Narrow Fan Beam (4.5º), GE Health Care, Madison, Wisconsin, USA).

2. V̇O2max measurement

   Time frame: 9 months

   Participants' V̇O2max was determined using an incremental exercise test on a cycle ergometer (COSMED E 200, Rome, Italy). The test consisted of 2 minutes of cycling at 60 W, 120 W, and 150 W, respectively. Afterward, the workload increased by 30 W every minute until voluntary exhaustion. Heart rate (HR) was continuously recorded during the test using a HR monitor. Breath-by-breath expired air was acquired throughout the test using an online gas analysis system (Quark Cardio Pulmonary Exercise Testing, COSMED, Rome, Italy). The gas analyzer was periodically calibrated according to the manufacturer's procedures prior to each test. The recorded value for V̇O2max was the highest achieved over a 30-second sampling period.

3. Measurement of resting metabolic rate (RMR)

   Time frame: 9 months

   RMR was determined using a breath-by-breath indirect system (CPET, Rome, Italy). Participants were required to fast for 10-12 hours before testing and limit physical activity on their way to the laboratory. Upon arriving at the laboratory, participants rested in a dimly lit, temperature-controlled room in a supine position, and were instructed to relax without falling asleep. The protocol involved a 20-minute rest in the supine position, followed by a 15-minute period of respiratory gas analysis. During the respiratory gas analysis, oxygen uptake and carbon dioxide output were measured using a breath-by-breath system with a breathing mask connected to a pre-calibrated computerized gas analyzer.

4. Calculation of resting substrate oxidation

   Time frame: 9 months

   We used the respiratory data (oxygen and carbon dioxide) collected during the RMR measurement to calculate resting fat (g/min) and resting carbohydrate oxidation (g/min) using the Frayn equation, as follows:

   Fat oxidation (g/min) = 1,67 × V̇O2 (L/min) - 1,67 × V̇CO2 (L/min) CHO oxidation (g/min) = 4,55 × V̇CO2 (L/min) - 3,21 × V̇O2 (L/min)

5. Measurement of substrate oxidation during submaximal exercise

   Time frame: 9 months

   A few minutes after RMR assessment, participants cycled for 30 minutes at a workload corresponding to 40% of their pre-determined V̇O2max on cycle ergometer (COSMED E 200, Italy) before and after the TRF program. The test commenced with a 3-minute warm-up at 60 W. V̇O2 and V̇CO2 during the test were recorded using an online gas analysis system (Quark Cardio Pulmonary Exercise Testing, COSMED, Rome, Italy). Fat oxidation and CHO oxidation were computed utilizing the equation proposed by Jeukendrup and Wallis.

6. Food Diary

   Time frame: 9 months

   All participants completed a 7-day food diary at the commencement of the study, during the third week, and at the end of the sixth week to assess any potential changes in participant dietary habits. An experienced dietician determined the portion size with household units, such as cups, pieces, or plates. In addition, the ingredients of mixed dishes were specified, and product name and standard weights of food items were used to calculate serving sizes. All the results were calculated and analyzed by the same experienced dietician using Nutrition Information System (BEBIS 6.1, Dr. J. Erhardt, Stutgart, Hohenheim, Germany).

7. Time Restricted Feeding

   Time frame: 9 months

   The TRF group received detailed nutrition education before the TRF program, and was instructed to follow the 16:8 program for six weeks, limiting their eating window to 8 hours daily (10:00 to 18:00 or alternatively 11:00 to 19:00), during which no calorie restriction was applied. During the 16-hour fasting window, the TRF group was asked to avoid calorie-containing foods and beverages. All participants in both groups were contacted twice a week to monitor dietary compliance in the TRF group and to maintain their existing eating habits in the control group.