Trial Review

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Trial registered on ANZCTR


Registration number
ACTRN12625001027404
Ethics application status
Approved
Date submitted
3/09/2025
Date registered
17/09/2025
Date last updated
17/09/2025
Date data sharing statement initially provided
17/09/2025
Type of registration
Prospectively registered

Titles & IDs
Public title
A Randomised Controlled Crossover Trial Comparing Two 3.5-Day Fasting Protocols (Water-Only vs. Vegetable-Based Fasting) on Short-Term Outcomes in Healthy Adults.
Scientific title
A Randomised Controlled Crossover Trial Comparing Two 3.5-Day Fasting Protocols (Water-Only vs. Vegetable-Based Fasting) on autophagy and other outcomes in Healthy Adults.
Secondary ID [1] 315295 0
None
Universal Trial Number (UTN)
Trial acronym
PROFASTA 2
Linked study record
his study is a substudy of the original PROFASTA trial (ACTRN12623000087651)

Health condition
Health condition(s) or problem(s) studied:
Obesity 338757 0
Metabolic Syndrome 338758 0
Condition category
Condition code
Diet and Nutrition 335054 335054 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 vegetable fasting diet (VFD) (intervention).

Arm 2 consists of a 3.5-day plant-based fasting protocol providing no more than 25% of their daily caloric intake, sourced exclusively from non-starchy raw or cooked vegetables. All meals will be professionally prepared by an expert chef to ensure consistency and adherence. The diet is intentionally very low in protein and carbohydrates, and strictly excludes grains, legumes, and added fats.

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 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 VFD meals will be collected from the clinic which will be prepared by the clinic Chef and comprised by the clinic Dietitian.
Intervention code [1] 331898 0
Lifestyle
Comparator / control treatment
Comparator: 3-day water-only fasting (intervention A), Only water is to be consumed (no food other liquids) in this arm,
Control group
Active

Outcomes
Primary outcome [1] 342677 0
Short-term change in autophagic flux in mononuclear cells (PBMCs) isolated from venous blood during fasting with or without the VFD Quantified by Western blot of PBMC samples. The change in autophagic flux will be compared between the two interventions (crossover design).
Timepoint [1] 342677 0
Change from baseline to day 1, day 2, day 3 (primary timepoint) after starting fast and after 4 days of refeeding (day 7).
Primary outcome [2] 342678 0
Short-term change in autophagic flux in PBMCs isolated from venous blood during 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] 342678 0
Change from baseline to day 1, day 2, day 3 (primary timepoint) after starting fast and after 3 days of refeeding (day 7).
Primary outcome [3] 342679 0
Medium-term change in autophagic flux in PBMCs isolated from venous blood during 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] 342679 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] 451782 0
Body weight (kg) measured by an electronic scale.
Timepoint [1] 451782 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] 451783 0
Waist circumference (cm) measured by a tape measure.
Timepoint [2] 451783 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] 451784 0
Body composition (body fat and fat free mass), measured by air displacement plethysmography via BOD POD.
Timepoint [3] 451784 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] 451785 0
Blood pressure, systolic and diastolic, measured by automated blood pressure monitor.
Timepoint [4] 451785 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] 451786 0
Peripheral capillary oxygen saturation (SPO2), measured by a pulse oximeter, together with blood pressure.
Timepoint [5] 451786 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] 451787 0
Flow-dependent vasodilation (FMD), measured by brachial arterial ultrasound technique.
Timepoint [6] 451787 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] 451788 0
Heart Rate Variability (HRV) measured by electrocardiography (ECG) at rest.
Timepoint [7] 451788 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] 451789 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] 451789 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] 451790 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] 451790 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] 451791 0
Habitual dietary intake, food diary and dietary recall will be recorded using Research Food Diary (Xyris).
Timepoint [10] 451791 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] 451792 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] 451792 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] 451793 0
Urinary F2-isoprostanes from 12h urine samples.
Timepoint [12] 451793 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] 451794 0
Microbiome composition of stool samples, assessed by 16S rRNA sequencing.
Timepoint [13] 451794 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] 451795 0
Capillary blood glucose measured either by finger prick electronic meter or continuous glucose monitor (Abbott).
Timepoint [14] 451795 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] 451796 0
Capillary blood ketones measured either by finger prick electronic meter or continuous glucose monitor (Abbott).
Timepoint [15] 451796 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] 451797 0
Fasting blood glucose measured from venous blood sampling.
Timepoint [16] 451797 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] 451798 0
Blood beta-hydroxy-butyrate measured from venous blood sampling.
Timepoint [17] 451798 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] 451799 0
Composite outcome of blood lipids (triglycerides, LDL-C, HDL-C) quantified by venous sampling.
Timepoint [18] 451799 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] 451800 0
Free fatty acids (FFAs) measured from venous blood sampling.
Timepoint [19] 451800 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] 451801 0
ApoB measured from venous blood sampling.
Timepoint [20] 451801 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] 451802 0
Lp(a) measured from venous blood sampling.
Timepoint [21] 451802 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] 451803 0
C-reactive protein (CRP) measured from venous blood sampling.
Timepoint [22] 451803 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] 451804 0
Blood lactate measured from venous blood sampling.
Timepoint [23] 451804 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] 451805 0
ALT (alanine transaminase) measured from venous blood sampling.
Timepoint [24] 451805 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] 451807 0
AST (aspartate transaminase) measured from venous blood sampling.
Timepoint [25] 451807 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] 451808 0
AlkPhos (alkaline phosphatase) measured from venous blood sampling.
Timepoint [26] 451808 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] 451809 0
Cortisol measured from venous blood plasma samples.
Timepoint [27] 451809 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] 451811 0
Free T3 hormone measured from venous blood plasma samples.
Timepoint [28] 451811 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] 451813 0
IGF-1 hormone measured from venous blood plasma samples.
Timepoint [29] 451813 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] 451814 0
FGF21 measured from venous blood plasma samples.
Timepoint [30] 451814 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] 451815 0
Insulin measured from venous blood plasma samples.
Timepoint [31] 451815 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] 451816 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] 451816 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] 451817 0
Composite outcome of changes in plasma proteome and metabolome, quantified from venous blood plasma samples. (exploratory outcome)
Timepoint [33] 451817 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] 451818 0
Composite outcome of immune function will be assessed by multiplex cytokine assay (Olink) from venous blood plasma samples. (exploratory outcome).
Timepoint [34] 451818 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] 451819 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] 451819 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] 451820 0
Short-term change in autophagic flux in colon mucosa biopsies during the fasting arms, Quantified by Western blot of colon mucosa samples. The change in autophagic flux will be compared between the two interventions (crossover design).
Timepoint [36] 451820 0
Change from baseline to day 3 after starting the fasting protocols.
Secondary outcome [37] 451821 0
Short-term change in autophagic flux in colon mucosa biopsies during 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] 451821 0
Change from baseline to day 3.
Secondary outcome [38] 451822 0
Medium-term change in autophagic flux in colon mucosa during 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] 451822 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
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 fasting interventions.
7. Participation in the parent study ACTRN12623000087651 is also an inclusion criterion for eligibility for inclusion in this study,
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 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. 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
Phase
Not Applicable
Type of endpoint/s
Safety/efficacy
Statistical methods / analysis
Changes over time with two bouts of intervention will also be assessed (pre-post design). This represents a non-randomised component of the trial. 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
22/09/2025
Actual
Date of last participant enrolment
Anticipated
31/07/2026
Actual
Date of last data collection
Anticipated
30/09/2026
Actual
Sample size
Target
22
Accrual to date
Final
Recruitment in Australia
Recruitment state(s)
NSW
Recruitment postcode(s) [1] 44639 0
2050 - Camperdown

Funding & Sponsors
Funding source category [1] 319867 0
Government body
Name [1] 319867 0
NHMRC
Country [1] 319867 0
Australia
Primary sponsor type
University
Name
University of Sydney
Address
Country
Australia
Secondary sponsor category [1] 322394 0
None
Name [1] 322394 0
Address [1] 322394 0
Country [1] 322394 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 318421 0
Sydney Local Health District Ethics Review Committee (RPAH Zone)
Ethics committee address [1] 318421 0
Ethics committee country [1] 318421 0
Australia
Date submitted for ethics approval [1] 318421 0
03/06/2025
Approval date [1] 318421 0
13/06/2025
Ethics approval number [1] 318421 0

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

Contacts
Principal investigator
Name 144134 0
Prof Luigi Fontana
Address 144134 0
Level 5 West D17 – Education and Research Hub The Charles Perkins Centre Camperdown The University of Sydney NSW 2006
Country 144134 0
Australia
Phone 144134 0
+61 02 8627 7499
Fax 144134 0
Email 144134 0
[email protected]
Contact person for public queries
Name 144135 0
Isabella de Ciutiis
Address 144135 0
Level 5 West D17 – Education and Research Hub The Charles Perkins Centre Camperdown The University of Sydney NSW 2006
Country 144135 0
Australia
Phone 144135 0
+61466691750
Fax 144135 0
Email 144135 0
[email protected]
Contact person for scientific queries
Name 144136 0
Isabella de Ciutiis
Address 144136 0
Level 5 West D17 – Education and Research Hub The Charles Perkins Centre Camperdown The University of Sydney NSW 2006
Country 144136 0
Australia
Phone 144136 0
+61466691750
Fax 144136 0
Email 144136 0
[email protected]

Data sharing statement
Will the study consider sharing individual participant data?
Yes
Will there be any conditions when requesting access to individual participant data?
Persons/groups eligible to request access:
• Researchers
Conditions for requesting access:
• Researchers can contact the PI if they have a research idea. The Principal Investigator will decide to release data based upon the proposal

What individual participant data might 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.

What types of analyses could be done with individual participant data?
• This will depend on the research proposal and discretion of the Principal Investigator

When can requests for individual participant data be made (start and end dates)?
From:
Data will be available after completion of the study (2025) and there will be no end date.

To:
Data will be available after completion of the study (2025) and there will be no end date.

Where can requests to access individual participant data be made, or data be obtained directly?
• Contact the Principal Investigator by email ([email protected])

Are there extra considerations when requesting access to individual participant data?
No


What supporting documents are/will be available?

No Supporting Document Provided


Results publications and other study-related documents

Documents added manually
No documents have been uploaded by study researchers.

Documents added automatically
No additional documents have been identified.