Trial Review

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Trial registered on ANZCTR


Registration number
ACTRN12623000087651
Ethics application status
Approved
Date submitted
11/01/2023
Date registered
25/01/2023
Date last updated
25/01/2023
Date data sharing statement initially provided
25/01/2023
Type of registration
Prospectively registered

Titles & IDs
Public title
Comparing the Effect of Two Prolonged Fasting Protocols on Short-term Outcomes in Healthy Individuals
Scientific title
A Randomised Controlled Crossover Trial to Evaluate the Effect of Two Prolonged Fasting Protocols on Autophagy in Healthy Individuals
Secondary ID [1] 307999 0
None
Universal Trial Number (UTN)
Trial acronym
PROFASTA
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Obesity 327668 0
Metabolic syndrome 327669 0
Condition category
Condition code
Diet and Nutrition 324752 324752 0 0
Obesity

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
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 min on a cycle ergometer. After 5 minutes rest period, participants will perform 6 repeated 1-min sprints at 90 rpm and 125% peak power output, separated by 1-min rest periods.
Intervention code [1] 324454 0
Lifestyle
Comparator / control treatment
Comparator: 3-day water-only fasting (intervention A)
Control group
Active

Outcomes
Primary outcome [1] 332573 0
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).
Timepoint [1] 332573 0
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).
Primary outcome [2] 333498 0
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).
Timepoint [2] 333498 0
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).
Primary outcome [3] 333499 0
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).
Timepoint [3] 333499 0
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).
Secondary outcome [1] 417322 0
Body weight (kg) measured by an electronic scale.
Timepoint [1] 417322 0
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.
Secondary outcome [2] 417323 0
Waist circumference (cm) measured by a tape measure.
Timepoint [2] 417323 0
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.
Secondary outcome [3] 417325 0
Body composition (body fat and fat free mass), measured by air displacement plethysmography via BOD POD.
Timepoint [3] 417325 0
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.
Secondary outcome [4] 417342 0
Blood pressure, systolic and diastolic, measured by automated blood pressure monitor.
Timepoint [4] 417342 0
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).
Secondary outcome [5] 417343 0
Peripheral capillary oxygen saturation (SPO2), measured by a pulse oximeter, together with blood pressure.
Timepoint [5] 417343 0
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).
Secondary outcome [6] 417344 0
Flow-dependent vasodilation (FMD), measured by brachial arterial ultrasound technique.
Timepoint [6] 417344 0
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.
Secondary outcome [7] 417345 0
Heart Rate Variability (HRV) measured by electrocardiography (ECG) at rest.
Timepoint [7] 417345 0
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.
Secondary outcome [8] 417347 0
Physical activity measured by a wrist worn Fitbit accelerometer device, including step count and heart rate. This will be assessed as a composite outcome.
Timepoint [8] 417347 0
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).
Secondary outcome [9] 417351 0
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.
Timepoint [9] 417351 0
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).
Secondary outcome [10] 417352 0
Habitual dietary intake, weighed food diary will be recorded using Research Food Diary (Xyris).
Timepoint [10] 417352 0
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).
Secondary outcome [11] 417353 0
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%).
Timepoint [11] 417353 0
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).
Secondary outcome [12] 417354 0
Urinary F2-isoprostanes from 12h urine samples.
Timepoint [12] 417354 0
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.
Secondary outcome [13] 417356 0
Microbiome composition of stool samples, assessed by 16S rRNA sequencing.
Timepoint [13] 417356 0
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.
Secondary outcome [14] 417357 0
Capillary blood glucose measured either by finger prick electronic meter or continuous glucose monitor (Abbott).
Timepoint [14] 417357 0
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).
Secondary outcome [15] 417359 0
Capillary blood ketones measured either by finger prick electronic meter or continuous glucose monitor (Abbott).
Timepoint [15] 417359 0
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).
Secondary outcome [16] 417360 0
Fasting blood glucose measured from venous blood sampling.
Timepoint [16] 417360 0
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).
Secondary outcome [17] 417361 0
Blood beta-hydroxy-butyrate measured from venous blood sampling.
Timepoint [17] 417361 0
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).
Secondary outcome [18] 417362 0
Composite outcome of blood lipids (triglycerides, LDL-C, HDL-C) quantified by venous sampling.
Timepoint [18] 417362 0
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).
Secondary outcome [19] 417368 0
Free fatty acids (FFAs) measured from venous blood sampling.
Timepoint [19] 417368 0
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).
Secondary outcome [20] 417369 0
ApoB measured from venous blood sampling.
Timepoint [20] 417369 0
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).
Secondary outcome [21] 417370 0
Lp(a) measured from venous blood sampling.
Timepoint [21] 417370 0
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).
Secondary outcome [22] 417371 0
C-reactive protein (CRP) measured from venous blood sampling.
Timepoint [22] 417371 0
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).
Secondary outcome [23] 417372 0
Blood lactate measured from venous blood sampling.
Timepoint [23] 417372 0
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).
Secondary outcome [24] 417373 0
ALT (alanine transaminase) measured from venous blood sampling.
Timepoint [24] 417373 0
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).
Secondary outcome [25] 417374 0
AST (aspartate transaminase) measured from venous blood sampling.
Timepoint [25] 417374 0
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).
Secondary outcome [26] 417375 0
AlkPhos (alkaline phosphatase) measured from venous blood sampling.
Timepoint [26] 417375 0
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).
Secondary outcome [27] 417376 0
Cortisol measured from venous blood plasma samples.
Timepoint [27] 417376 0
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).
Secondary outcome [28] 417377 0
Free T3 hormone measured from venous blood plasma samples.
Timepoint [28] 417377 0
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).
Secondary outcome [29] 417378 0
IGF-1 hormone measured from venous blood plasma samples.
Timepoint [29] 417378 0
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).
Secondary outcome [30] 417379 0
FGF21 measured from venous blood plasma samples.
Timepoint [30] 417379 0
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).
Secondary outcome [31] 417380 0
Insulin measured from venous blood plasma samples.
Timepoint [31] 417380 0
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).
Secondary outcome [32] 417381 0
Composite outcome of Pheno-Age clock will be estimated by a published formula from standard blood parameters, measured from venous blood sampling. (exploratory outcome)
Timepoint [32] 417381 0
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).
Secondary outcome [33] 417382 0
Composite outcome of changes in plasma proteome and metabolome, quantified from venous blood plasma samples. (exploratory outcome)
Timepoint [33] 417382 0
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).
Secondary outcome [34] 417383 0
Composite outcome of immune function will be assessed by multiplex cytokine assay (Olink) from venous blood plasma samples. (exploratory outcome)
Timepoint [34] 417383 0
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).
Secondary outcome [35] 417385 0
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)
Timepoint [35] 417385 0
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.
Secondary outcome [36] 417804 0
Short-term change in autophagic flux in 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 flux will be compared between the two interventions (crossover design).
Timepoint [36] 417804 0
Change from baseline to day 3 after starting water-only fasting.
Secondary outcome [37] 417805 0
Short-term change in autophagic flux in colon mucosa biopsies during water-only fasting, quantified by Western blot of colon mucosa samples. The change in autophagic flux will be compared by pooling both interventions, in time (pre-post design).
Timepoint [37] 417805 0
Change from baseline to day 3 after starting water-only fast.
Secondary outcome [38] 417810 0
Medium-term change in autophagic flux in colon mucosa during water-only fasting, quantified by Western blot of colon mucosa samples. The change in autophagic flux will be compared by pooling both interventions, in time (pre-post design).
Timepoint [38] 417810 0
Change from baseline to day 3 of first fasting period and month 2 follow-up.

Eligibility
Key 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.
Minimum age
18 Years
Maximum age
70 Years
Sex
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
1. Participants that are pregnant, breastfeeding or of childbearing potential who are not willing to avoid becoming pregnant during the study.
2. History of chronic disease processes that according to the investigators could interfere with ability to undergo exercise tests fasting or may affect the interpretation of results.
3. People with comorbidities such as (but not limited to) hypertension, diabetes, diagnosed cardiovascular disease
4. History of supplement and medication use that may affect the interpretation of results.
5. Health condition dietary requirements or life situation that would interfere with study participation and compliance.
6. Unintentional weight loss (>10% body weight) over the past 5 years, smoking, psychiatric or behavioural problems (history of drug and alcohol abuse, eating disorder).
7. Inability to perform cycling exercise: incremental peak power test during the baseline assessment visit, or the 90-minute glycogen-depleting exercise at the start of the fasting intervention.
8. People judged at the investigator’s discretion as being unsuitable to participate.

Study design
Purpose of the study
Educational / counselling / training
Allocation to intervention
Randomised controlled trial
Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)
Participants will be randomised using computer generated random numbers, via a web-based randomisation system. The statistician will design the randomisation schedule, provided to the research co-ordinator on a per-participant basis, at the time of randomisation.
Methods used to generate the sequence in which subjects will be randomised (sequence generation)
Participant allocation to Arm 1 or Arm 2 will be carried out by variable block randomisation with age stratification via web based randomiser validated by statistician.
Masking / blinding
Open (masking not used)
Who is / are masked / blinded?



Intervention assignment
Crossover
Other design features
Changes over time with two bouts of intervention will be also be assessed (pre-post design). This represents a non-randomised component of the trial.
Phase
Not Applicable
Type of endpoint/s
Safety/efficacy
Statistical methods / analysis
Sample size calculation used previously published data on autophagic flux quantification by Western blot of LC3B-II protein. Change in LC3B-II to beta-actin (loading control) ratio to estimate sample size; using ANOVA, repeated measures design, a standard deviation of 0.4328, with 80% power and alpha of 0.05, a difference of 25% (control group mean = 1.36) may be detected with a sample size of 15 per group. We have estimated 25% drop-out rate thus resulting in starting sample of 20.

Analysis plan. Analysis of measures collected at multiple timepoints will be performed using ANOVA, repeated measures design and/or linear mixed model regression with and without covariates to test the effect of the 2 different fasting interventions. Linear regression and/or general additive model will be used to test the relationship between habitual dietary intakes and baseline measures and separately, the magnitude of response to fasting. In the exploratory analysis, correlation of autophagic flux readout with quantified molecular multi-omic biomarkers will be assessed to identify autophagic activity biomarker candidates for future studies.

Recruitment
Recruitment status
Not yet recruiting
Date of first participant enrolment
Anticipated
1/03/2023
Actual
Date of last participant enrolment
Anticipated
1/06/2024
Actual
Date of last data collection
Anticipated
1/09/2024
Actual
Sample size
Target
20
Accrual to date
Final
Recruitment in Australia
Recruitment state(s)
NSW
Recruitment hospital [1] 23792 0
Royal Prince Alfred Hospital - Camperdown
Recruitment postcode(s) [1] 39241 0
2050 - Camperdown

Funding & Sponsors
Funding source category [1] 312264 0
Government body
Name [1] 312264 0
NHMRC
Country [1] 312264 0
Australia
Primary sponsor type
University
Name
University of Sydney
Address
City Road,
Camperdown NSW 2006
Country
Australia
Secondary sponsor category [1] 313804 0
None
Name [1] 313804 0
Address [1] 313804 0
Country [1] 313804 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 311637 0
Sydney Local Health District Ethics Review Committee (RPAH Zone)
Ethics committee address [1] 311637 0
Ethics committee country [1] 311637 0
Australia
Date submitted for ethics approval [1] 311637 0
12/04/2022
Approval date [1] 311637 0
20/06/2022
Ethics approval number [1] 311637 0
X22-0110

Summary
Brief summary
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 121806 0
Prof Luigi Fontana
Address 121806 0
Level 5 West
D17 – Education and Research Hub
The Charles Perkins Centre
Camperdown
The University of Sydney
NSW
2006
Country 121806 0
Australia
Phone 121806 0
+61 2 8627 7499
Fax 121806 0
Email 121806 0
[email protected]
Contact person for public queries
Name 121807 0
Andrius Masedunskas
Address 121807 0
Level 5 West
D17 – Education and Research Hub
The Charles Perkins Centre
Camperdown
The University of Sydney
NSW
2006
Country 121807 0
Australia
Phone 121807 0
+61 4 6634 6538
Fax 121807 0
Email 121807 0
[email protected]
Contact person for scientific queries
Name 121808 0
Andrius Masedunskas
Address 121808 0
Level 5 West
D17 – Education and Research Hub
The Charles Perkins Centre
Camperdown
The University of Sydney
NSW
2006
Country 121808 0
Australia
Phone 121808 0
+61 4 6634 6538
Fax 121808 0
Email 121808 0
[email protected]

Data sharing statement
Will individual participant data (IPD) for this trial be available (including data dictionaries)?
Yes
What data in particular will be shared?
Non-identifiable participant data will be shared based upon the research proposal and subsequent discretion of the Principal Investigator. This will include all data.
When will data be available (start and end dates)?
Data will be available after completion of the study (2025) and there will be no end date.
Available to whom?
Researchers can contact the PI if they have a research idea. The Principal Investigator will decide to release data based upon the proposal.
Available for what types of analyses?
This will depend on the research proposal and discretion of the Principal Investigator
How or where can data be obtained?
Contact the Principal Investigator by email ([email protected])


What supporting documents are/will be available?

No Supporting Document Provided



Results publications and other study-related documents

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