Comparing the Effect of Two Prolonged Fasting Protocols on Short-term Outcomes in Healthy Individuals

INTERVENTION: Arm 1: 3‐day water‐only fasting (comparator) Arm 2: 3‐day water‐only fasting with one bout of glycogen‐lowering cycling exercise at the beginning of the fast (intervention). After completion of the intervention/comparator, and following the 4‐week washout period, the two arms will be crossed over, and the intervention/comparator will be repeated. After 4 week wash out period, and before the second period of fasting, "Month 1" timepoint samples and data will be collected (baseline #2). Finally, 4 weeks after completion of the second bout of fasting, "Month 2" follow up timepoint samples and data will be collected. Adherence to the fasting intervention will be monitored via the weighted food diary and blood glucose and ketone monitoring. After each fasting period, participants will revert to their usual diets gradually, over the refeeding period of 4 days. The participants will be advised to start with low carbohydrate vegetable soup as their first meal and to consume a low carbohydrate vegetarian diet consisting of vegetables, salads, soups, and fruit for the first day. Participants will be advised to start with small portions and increase portion size gradually, returning to unrestricted/usual diets during the 4 day refeeding period. The diet will not be imposed or controlled, however, it will be documented via 4‐day weighted food diary. Fasting will be carried out at home with daily clinic visit each morning. Adherence will be tracked by daily questionnaires, blood glucose and ketone measurements. The glycogen‐lowering cycling exercise will be performed at the clinic on the first day of fasting. Participants will cycle at 70 rpm, constant power output, at an intensity corresponding to 70% of their previously determined peak power output for 60 CONDITION: Diet and Nutrition ‐ Obesity Obesity;Metabolic syndrome; ; Obesity ; Metabolic syndrome PRIMARY OUTCOME: Medium‐term change in autophagic flux in PBMCs isolated from venous blood during water‐only fasting, quantified by Western blot of PBMC samples. The change in autophagic flux will be compared by pooling both interventions, in time (pre‐post design).; [Change from baseline to day 3 of first fasting period, month 1 (baseline#2, before the second fasting period), day 3 of second fasting period and month 2 which is a follow‐up after second fasting period (primary timepoint). ] Short‐term change in autophagic flux in mononuclear cells (PBMCs) isolated from venous blood during water‐only fasting with or without one bout of glycogen depleting exercise, quantified by Western blot of PBMC samples. The change in autophagic flux will be compared between the two interventions (crossover design).[Change from baseline to day 1, day 2, day 3 (primary timepoint) after starting water‐only fast and after and after 4 days of refeeding (day 7).] Short‐term change in autophagic flux in PBMCs isolated from venous blood during water‐only fasting, quantified by Western blot of PBMC samples. The change in autophagic flux will be compared by pooling both interventions, in time (pre‐post design).[Change from baseline to day 1, day 2, day 3 (primary timepoint) after starting water‐only fast and after and after 3 days of refeeding (day 7). ] SECONDARY OUTCOME: AlkPhos (alkaline phosphatase) measured from venous blood sampling.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] ALT (alanine transaminase) measured from venous blood sampling.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] ApoB measured from venous blood sampling.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] AST (aspartate transaminase) measured from venous blood sampling.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Blood beta‐hydroxy‐butyrate measured from venous blood sampling. [Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Blood lactate measured from venous blood sampling.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Blood pressure, systolic and diastolic, measured by automated blood pressure monitor.[Change from baseline to day 1, day 2, day 3 of first fasting period, day 7, month 1 (baseline#2, before the second fasting period), day 1, day 2, day 3 of second fasting period, day 7, and month 2 which is a follow‐up after second fasting period (the main secondary timepoint).] Body composition (body fat and fat free mass), measured by air displacement plethysmography via BOD POD.[Change from baseline to day 3 of first fasting period, day 7, month 1 (baseline#2, before the second fasting period), day 3 of second fasting period, day 7 and month 2 which is a follow‐up after second fasting period.] Body weight (kg) measured by an electronic scale.[Change from baseline to day 1, day 2, day 3 of first fasting period, day 7, month 1 (baseline#2, before the second fasting period), day 1, day 2, day 3 of second fasting period, day 7 (end of the second refeeding period), and month 2 which is a follow‐up after second fasting period.] C‐reactive protein (CRP) measured from venous blood sampling.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Capillary blood glucose measured either by finger prick electronic meter or continuous glucose monitor (Abbott).[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Capillary blood ketones measured either by finger prick electronic meter or continuous glucose monitor (Abbott).[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Composite outcome of blood lipids (triglycerides, LDL‐C, HDL‐C) quantified by venous sampling.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Composite outcome of changes in plasma proteome and metabolome, quantified from venous blood plasma samples. (exploratory outcome)[Measured on the day before fasting (baseline) and at the end of fasting and refeeding, for both periods, month 1, and at the follow‐up timepoint (month 2).] Composite outcome of expression profiles changes will be assessed by single cell RNA sequencing (scRNAseq) in PBMCs, buccal mucosa cells and colon mucosa samples. (exploratory outcome)[Measured on the day before fasting (baseline) and at the end of fasting and refeeding, for both periods, month 1 (baseline#2, before the second fasting period), and at the follow‐up timepoint (month 2). Note: for colon mucosa samples, only three timepoints will be tested: baseline, day 3 of first fasting period and month 2 follow‐up.] Composite outcome of immune function will be assessed by multiple Xcytokine assay (Olink) from venous blood plasma samples. (exploratory outcome)[Measured on the day before fasting (baseline) and at the end of fasting and refeeding, for both periods, month 1, and at the follow‐up timepoint (month 2).] Composite outcome of Pheno‐Age clock will be estimated by a published formula from standard blood parameters, measured from venous blood sampling. (exploratory outcome)[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Cortisol measured from venous blood plasma samples.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Fasting blood glucose measured from venous blood sampling. [Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] FGF21 measured from venous blood plasma samples.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Flow‐dependent vasodilation (FMD), measured by brachial arterial ultrasound technique.[Change from baseline to day 3 of first fasting period, day 7, month 1 (baseline#2, before the second fasting period), day 3 of second fasting period, day 7 and month 2 which is a follow‐up after second fasting period.] Free fatty acids (FFAs) measured from venous blood sampling.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Free T3 hormone measured from venous blood plasma samples.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Habitual dietary intake, weighed food diary will be recorded using Research Food Diary (Xyris).[Data will be recorded over 5 days before the first fasting period (baseline), during the first fasting (3 days) and refeeding period (4 days), 5 days before the second fasting period (month 1), during the second fasting (3 days) and refeeding period (4 days), and 5 days before the follow‐up timepoint (month 2).] Heart Rate Variability (HRV) measured by electrocardiography (ECG) at rest.[Change from baseline to day 3 of first fasting period, day 7, month 1 (baseline#2, before the second fasting period), day 3 of second fasting period, day 7 and month 2 which is a follow‐up after second fasting period.] IGF‐1 hormone measured from venous blood plasma samples.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Insulin measured from venous blood plasma samples.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Lp(a) measured from venous blood sampling.[Measured on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Medium‐term change in autophagic flu Xin colon mucosa during water‐only fasting, quantified by Western blot of colon mucosa samples. The change in autophagic flu Xwill be compared by pooling both interventions, in time (pre‐post design).[Change from baseline to day 3 of first fasting period and month 2 follow‐up.] Microbiome composition of stool samples, assessed by 16S rRNA sequencing.[Measured in samples collected at baseline, day 3 of first fasting period, day 7, month 1 (baseline#2, before the second fasting period), day 3 of second fasting period, day 7 and month 2 which is a follow‐up after second fasting period.] Peripheral capillary oxygen saturation (SPO2), measured by a pulse oximeter, together with blood pressure.[Change from baseline to day 1, day 2, day 3 of first fasting period, day 7, month 1 (baseline#2, before the second fasting period), day 1, day 2, day 3 of second fasting period, day 7, and month 2 which is a follow‐up after second fasting period (the main secondary timepoint).] Physical activity measured by a wrist worn Fitbit accelerometer device, including step count and heart rate. This will be assessed as a composite outcome.[Measurements will be taken over 7 days before the first fasting period (baseline), during the first fasting (3 days) and refeeding period (4 days), 7 days before the second fasting period (month 1), during the second fasting (3 days) and refeeding period (4 days), and 7 days before the follow‐up timepoint (month 2).] Short‐term change in autophagic flu Xin colon mucosa biopsies during water‐only fasting with or without one bout of glycogen depleting exercise, quantified by Western blot of colon mucosa samples. The change in autophagic flu Xwill be compared between the two interventions (crossover design).[Change from baseline to day 3 after starting water‐only fasting.] Short‐term change in autophagic flu Xin colon mucosa biopsies during water‐only fasting, quantified by Western blot of colon mucosa samples. The change in autophagic flu Xwill be compared by pooling both interventions, in time (pre‐post design).[Change from baseline to day 3 after starting water‐only fast.] Sleep duration and quality measured by a wrist worn Fitbit accelerometer device, including sleep efficiency, duration of sleep stages, resting heart rate and heart rate variability during sleep. This will be assessed as a composite outcome.[Measurements will be taken over 7 days before the first fasting period (baseline), during the first fasting (3 days) and refeeding period (4 days), 7 days before the second fasting period (month 1), during the second fasting (3 days) and refeeding period (4 days), and 7 days before the follow‐up timepoint (month 2).] Symptoms/adverse events assessed by a questionnaire, custom, unvalidated. The most common reported symptoms during the fasting are sleep disturbances, headaches, weakness, perception of hunger while adverse events are extremely rare (less than 1%).[Questionnaire will be recorded on the day before fasting (baseline) and during each day of fasting and refeeding, for both periods, and at the follow‐up timepoint (month 2).] Urinary F2‐isoprostanes from 12h urine samples.[Measured in samples collected at baseline, day 3 of first fasting period, day 7, month 1 (baseline#2, before the second fasting period), day 3 of second fasting period, day 7 and month 2 which is a follow‐up after second fasting period.] Waist circumference (cm) measured by a tape measure.[Change from baseline to day 1, day 2, day 3 of first fasting period, day 7, month 1 (baseline#2, before the second fasting period), day 1, day 2, day 3 of second fasting period, day 7, and month 2 which is a follow‐up after second fasting period.] INCLUSION CRITERIA: 1. Disease status/ or disease group for study: healthy, without disability that may impede cycling exercise. 2. Sex: male and female 3. Age range: 18‐70 years old 4. BMI range: 20‐40kg/m2 5. Basic written and spoken English language proficiency needed for the completion of the questionnaires. 6. Willing and able to undergo all study evaluations and adhere to exercise and fasting interventions.