Physiological and Sensory Responses to Prolonged Fasting in Humans
Phase: NA
Conditions: Fasting, Metabolic Disturbance, Energy Supply; Deficiency, Adiposity
Sponsor: University of Bath

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Version 2.1; 23/09/20 
 
PROTOCOL 
Physiological and sensory responses to prolonged fasting in humans 
REACH:  
Version 2.1 23/09/2020 
 
 
 
 
 
 
 
 
 
 
 
 
Investigators 
Mr Harry Smith – PhD student, Dept. for Health, University of Bath 
Mr Jonathan Watkins – PhD student, Dept. for Health, University of Bath 
Mr Aaron Hengist – PhD student, Dept. for Health, University of Bath 
Dr Jean-Philippe Walhin – Lecturer, Dept. for Health, University of Bath 
Dr Jen Maher – Researcher, Dept. for Health, University of Bath 
Dr James Turner – Senior Lecturer, Dept. for Health, University of Bath 
Dr Javier Gonzalez – Reader, Dept. for Health, University of Bath 
Prof Kostas Tsintzas – Professor, School of Life Sciences, University of Nottingham 
Prof Christopher Eccleston– Professor, Dept. for Health, University of Bath 
Prof Dylan Thompson – Professor, Dept. for Health, University of Bath 
Prof James Betts – Professor, Dept. for Health, University of Bath 
 
Address 
1 West 3.102 
Department for Health 
University of Bath 
Claverton Down 
Bath 
BA2 7AY 
 
Contact information 
Mr Harry Smith 
 
email: H.A.Smith@bath.ac.uk           Tel: 01225 383731 
Prof James Betts 
 
email: J.Betts@bath.ac.uk  
           Tel: 01225 383448 
 
 
1. BACKGROUND 
The human genome has evolved around periods of restricted food intake and is highly 
adapted with the relevant pathways to cope with this. Yet, there are still gaps in our 
understanding of these physiological responses. In the absence of energy intake, the body 

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maintains life by using stored energy, which is sufficient to sustain life over a prolonged 
period (> 1 year) if energy stores are sufficient [1]. In particular, the shift in metabolic 
substrate initially acts to maintain glucose before then acting to try and spare muscle tissue. 
However, there is also a significant shift in amino acid metabolism, and little research has 
explored this in human skeletal muscle and or adipose tissue. Furthermore, to the best of 
our knowledge, little or no research has investigated changes in the genes/proteins that 
regulate lipid/glucose metabolism in adipose tissue in response to prolonged fasting. 
 
Furthermore, it has been established that periods of prolonged fasting result in a reduction in 
skeletal muscle insulin sensitivity [2], perhaps due to the excess of fatty acids resultant from 
severe energy deficit. However, this has primarily been established using proof of principle 
methods (e.g. oral, or intravenous tolerance tests). Whilst these have been invaluable in 
establishing effects, it is also important to employ more ecologically valid models, such as 
mixed meal tolerance tests. 
 
 
2. OVERVIEW OF PROPOSED RESEARCH 
 
2.1 Hypothesis 
Due to the exploratory/novel nature of the model this is not considered hypothesis driven 
research.  
 
2.2 Aims and Objectives 
Aims 
1. To establish the metabolic/molecular response in both adipose tissue and skeletal muscle 
to acute starvation (~82 h). 
2. To establish the sensory experience to prolonged fasting (pain, fatigue, drive). 
 
Objectives 
Assess Amino acids in adipose, skeletal muscle and circulation in response to prolonged 
fasting 
Assess postprandial response to a meal pre and post prolonged fast 
Assess anthropometric changes in response to prolonged fasting 
Assess the sensory experience in response to prolonged fasting 
 
2.3 Rationale 
We plan to study participants before, after and during ~82 hours of no energy intake. Whilst 
few studies have extended starvation beyond 48-72 h, this duration is sufficient to observe 
the established shift towards greater reliance on lipids to fuel metabolism [2-5]. Necessarily, 
extension of the starvation beyond this duration therefore guarantees stability in the reliance 
on endogenous fuels (e.g. NEFAs, and ketones). At the level of the adipose tissue, an 
increase in the efflux of NEFA from adipose tissue as well as a reduction in glucose uptake 
is apparent during the early stages of a prolonged fast (~20 h) [5-7]. Necessarily, given that 
changes in metabolism within the adipose tissue occur this rapidly, it is surprising that so few 
studies have included measures of this tissue under conditions of prolonged fasting. In 
particular, assessment of genes and proteins responsible for regulating substrate 
metabolism in the adipose tissue would provide interesting insight into the metabolic 
flexibility of this tissue under severe energy deficit. Furthermore, measurement of amino 
acids and their respective ketoacids in adipose, skeletal muscle, and the circulation would 
further our understanding of how these metabolically important tissues respond in order to 
preserve/maintain skeletal muscle during energy deficit [2, 8]. 

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Surprisingly, only one study to date has investigated changes in post-prandial metabolism 
following mixed meal ingestion, as opposed to oral or intravenous tolerance tests [2, 9, 10]. 
Notably, whilst this study aimed to achieve a 72-h starve, provision of the mixed meal 
tolerance test immediately prior to beginning the 72-h starvation period resulted in an actual 
starve duration closer to ~2 days. Given the dramatic effect of acute starvation on gastric 
emptying [9] it is especially important to investigate the meal responses. Furthermore, given 
that insulin resistance following prolonged fasting persists even following a 24 h refeed, it 
would be insightful to include measures of sequential meals (i.e. the response to the 
standardised lunch and dinner will be assessed via continual glucose monitors).  
Finally, few, if any, studies have assessed the physical activity response to periods of 
prolonged fasting. Necessarily, methods such as doubly labelled water would not be 
appropriate due to the underlying assumptions with RQ as well as only giving a total energy 
expenditure value. Alternatively, employment of combined heart rate/accelerometery 
(Actiheart), would be appropriate in order to assess the change in physical activity levels, 
whilst giving a detailed breakdown in changes in intensity. 
 
2.4 Study design 
This study will employ a single-arm, non-randomised design in which participants will act as 
their own control prior to undergoing 3 days of starvation. The control phase includes ~3 
days of habitual energy balance assessment before coming into the laboratory for baseline 
metabolic and anthropometric assessment. Participants will be provided with monitoring 
equipment throughout the 3 days of starvation and will be asked to provide a blood sample 
as well as record their blood pressure, weight, and urinary ketones each day. On the middle 
day participants will also be asked to come into the lab to provide a blood sample at 2000 h. 
 
 
 
 
Figure 1. Protocol schematic depicting the study design. 
 
 

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2.5 Outcome measures 
Primary 
Metabolic response to feeding before and after a prolonged fast 
Change in amino acid metabolism (i.e. BCAAs and ketoacids) in skeletal muscle, adipose 
tissue, and in the circulation 
 
 
Secondary 
Change in circulating glycerol and FFAs across the prolonged fast (indices of lipolysis from 
adipose tissue)  
Change in Autophagy (in skeletal muscle and neutrophils) 
Total nitrogen (or at least urea nitrogen) in urine (24h collection) throughout the fast (or at 
least 1st vs last day)  
Change in plasma lactate and pyruvate in response to prolonged fasting (indices of Cori 
cycle) 
Change in urinary (acetoacetate), and circulating (beta-hydroxybutyrate) ketones in 
response to prolonged fasting. 
Change in body mass, pre-post, and during the prolonged fast. 
Change in blood pressure pre-post, and during the prolonged fast. 
Change in waist-hip circumferences pre-post the prolonged fast. 
Change in sensory experience (pain, fatigue, drive) in response to prolonged fasting.  
Change in haematocrit/haemoglobin pre-post the prolonged fast. 
Change in grip strength pre-post the prolonged fast. 
Change in reaction time pre-post the prolonged fast. 
Change in aspects of immune function and infection risk 
Change in interstitial fluid glucose concentrations pre-, post-, and during the prolonged fast 
(assessed via continual glucose monitoring)  
Occurrence of adverse events (e.g. nausea, vomiting, migraines) 
 
2.6 Statistical analysis  
Sample estimate:  
Due to the drastic nature of the intervention outlined (i.e. the complete abstinence from 
energy intake), a relatively small sample size will be required. As such, based on previous 
research, a sample size of 6-10 participants should be sufficient to observe changes in 
metabolism with this duration of fast. 
 
Descriptive statistics will be calculated using Microsoft Excel. Normality of data will be 
assessed using GraphPad Prism 8.0.  Repeated measures ANOVA Will be employed to 
identify significant effects of TREATMENT & TIME for post-prandial data. Paired t-test or 
Wilcoxon will be used to compare variables assessed only pre-and post-intervention. One-
way ANOVA will be used to assess changes in variables assessed each day. Bonferroni 
post-hoc tests will locate significant differences where they exist. Normalised confidence 
intervals will be calculated to plot as error bars on figures. The magnitude of these CIs 
directly infers the difference between means at each time point (i.e. statistical significance) 
rather than variance of individual values around the mean. 
 
3. Recruitment  
Principally this study will recruit from within the department of health however of needed then 
participants will be recruited from the University of Bath campus a by word of mouth and by 
poster advertisement. The full enrolment protocol is outlined below, however participation 

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will be centred on the provision of obtaining informed consent prior the study and throughout 
participation.  
 
Inclusion criteria 
• 
Body mass index 20.0-29.9 kg∙m-2  
• 
Age 18-45 years 
• 
Willing to abstain from food intake for just over 3 days (~82 hours) 
• 
Able and willing to provide informed consent and safely comply with study procedures 
• 
Females to maintain record of regular menstrual cycle phase or contraceptive use 
• 
No anticipated changes in lifestyle during the study (e.g. holidays or exercise 
programmes) 
 
Exclusion criteria 
• 
Any reported condition or behaviour deemed either to pose undue personal risk to the 
participant or introduce bias 
• 
Any diagnosed metabolic disease (e.g. type 1 or type 2 diabetes) 
• 
Any reported use of substances which may pose undue personal risk to the participants 
or introduce bias into the experiment 
• 
Pregnancy 
• 
Breastfeeding 
• 
Lifestyle not conforming to standard sleep-wake cycle (e.g. shift worker) 
• 
History of eating disorders (e.g. anorexia) 
• 
Any reported recent (<6 months) change in body mass (± 3%) 
• 
Uncontrolled hyperthyroidism 
• 
Advanced cerebrovascular insufficiency or dementia 
• 
Advanced liver or kidney insufficiency 
• 
History of migraine and or headache 
• 
Psychotic disorders 
• 
Unstable or severe coronary artery disease 
• 
Retinal detachment 
• 
Duodenal or stomach ulcer 
• 
Cancer and malignant disease 
• 
Regular use of Non-steroidal anti-inflammatory drugs (NSAIDs) 
• 
Use of Systemic corticoids 
• 
Use of Antihypertensives (especially beta-blockers and diuretics) 
• 
Use of Antidiabetics 
• 
Use of Anti-coagulants 
• 
Use of Psychotropics (especially neuroleptics and lithium) 
• 
Use of Anticonvulsants. 
 
 
Participants will provide informed consent both verbally and in written form. Upon expression 
of initial interest in the study, potential participants will be provided with the relevant 
appended ‘Participant information sheet’ to read in their own time, dependent on which 
phase(s) of the study they wish to take part in. Should they then decide they would like to 
participate in the study, they will be invited to a pre-enrolment meeting at the University, 
whereby a member of the research team, who is experienced in taking informed consent, will 
explain the study protocols and procedures. Potential participants are encouraged to ask any 
questions they may have, in order for them to fully understand what participating in the study 

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entails. Following this, if the individual is still willing to participate, they will fill out 2 copies of 
the appended ‘Informed Consent Form’ - – (V1.0 18/08/20), and eligibility screening will 
occur. 
 
3. Confidentiality  
Each participant will be allocated a unique study identification code upon enrolment to the 
study. This will be noted alongside their contact information in a designated file which will be 
kept locked in a separate office. All subsequent documents used throughout the study will be 
marked with this identification code rather than any identifiable information to prevent any 
association between personal data and recorded outcome measures by any unauthorised 
personnel or in any presentation of the data. Any electronic identifiable information will be 
kept in a password protected computer in a locked office of which only the members of the 
research team can access. Additionally, no identifiable information will be included in the 
presentation and dissemination of the results of this study.   
 
4. Trial days 
3 days prior to the first experimental visit participants will visit the laboratory to be fitted with 
a continual glucose monitor, an actiheart activity monitor, as well as a food diary and food 
scales. They will be asked to record energy intake and to standardise physical activity, 
caffeine, and alcohol intake. 
 
Upon finishing the 3 days of lifestyle standardisation participants will enter the laboratory for 
where weight, height, waist:hip circumference, sagittal-abdominal diameter, and skin-fold will 
be assessed prior to assessment of resting metabolic rate, blood pressure and urinary 
ketones. Participants will also undergo grip strength, reaction time, and sensory experience 
assessment. Following this, samples of skeletal muscle and adipose tissue will be collected 
from the vastus lateralis and abdominal adipose tissue respectively. An intravenous cannula 
will then be placed a 10 mL blood samples will be drawn before ingestion of a carbohydrate 
rich porridge breakfast (7.3 mg carbohydratekJ-1). 
 
A 3-hour postprandial period follows the ingestion of breakfast with blood samples will be 
collected at 15, 30- and 60-minute intervals during the first, second and third hour 
respectively alongside hourly expired gas samples and assessment of subjective appetite 
and mood. Upon completion of the 3-hour post-breakfast period, participants will have 
completed the visit and will be provided with a standardised lunch and dinner. Participants 
will also be provided with urinary ketone sticks, a blood pressure monitor, a set of weighing 
scales, as well as an actiheart monitor.  
 
Participants will be asked to assess urinary ketones using provided assessment sticks in the 
evening at 2000 h.  
 
5. Intervention  
Participants will be provided with their last meal at ~1900 h on the day of the meal testing. 
From this point onwards, participants will be asked to refrain from eating for ~82 hours until 
their return visit to the laboratory. Throughout this period of extended fasting participants will 
be provided with 80 mmol sodium and 40mmol potassium daily (to consume in the form of 
slow-release tablets) to minimize the confounding effects of fluid deprivation and 
intravascular volume depletion on cardiovascular reflexes and sympathetic nervous system 
activity. Subjects will also be permitted to consume water, non-sugared carbonated drinks 

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and, with the exception of the day of the pre/post measurements, non-sugared decaffeinated 
coffee and tea (if they normally consume tea and coffee). 
 
Upon waking each day participants will be asked to assess their urinary ketone levels, blood 
pressure, and weight. Participants will be asked to repeat this in the evening at 2000 h.  
 
Each day participants will be asked to return to the laboratory to provide a blood sample. On 
the second day of starvation participants will also be asked to provide a blood sample at 
2000 h. 
 
Furthermore, throughout the intervention participants will be asked to wear an actiheart 
monitor in order to assess physical activity levels in response to prolonged fasting.  
 
6. Follow up visit 
Upon completion of the intervention, participants will visit the laboratory at the University of 
Bath, and repeat the pre-intervention visit with the porridge breakfast. Following this 
repeated visit, participants will again be provided with a standardised lunch and dinner. 
 
 
7. Ethical considerations of risk and discomfort 
Acute starvation may cause discomfort for participants including headaches and lack of 
energy. All participants will be supervised by a member of the research team throughout the 
protocol. Participants who drop out after one day of fasting will not be included in follow up 
testing, but will be provided with food to break their fast. Conversely, participants who decide 
to not continue with study following 2 days of fasting will be asked to come in for follow up 
testing/standardised meal provision. Biopsies also introduce a risk for discomfort for 
participants but will only be performed by trained and experienced members of staff who 
follow the guidelines for safe practice. Intravenous cannulation has the potential to cause 
minor discomfort and bruising. Furthermore, there is a small associated risk of infection 
and/or embolism, where blood flow to the vein becomes partially restricted. In order to 
minimise these risks, cannulation will only be carried out by qualified and experienced staff 
members. The cannula is an essential aspect will only be in the vein for a maximum of 3 
hours per laboratory visit, whereas the venepuncture needle on days 1,2, and 3 of the fast 
will be removed as soon as the sample is obtained. 
 
8. Benefits to participants  
Participants will be provided with standardised lunches as well as dinners on the pre and 
post intervention days.  
 
 
References 
 
1. 
Stewart WK, Fleming LW. Features of a successful therapeutic fast of 382 days' 
duration. Postgrad Med J. 1973 Mar;49(569):203-9. 
2. 
Tsintzas K, Jewell K, Kamran M, Laithwaite D, Boonsong T, Littlewood J, et al. 
Differential regulation of metabolic genes in skeletal muscle during starvation and refeeding 
in humans. J Physiol. 2006 Aug 15;575(Pt 1):291-303. 
3. 
Romijn JA, Godfried MH, Hommes MJ, Endert E, Sauerwein HP. Decreased glucose 
oxidation during short-term starvation. Metabolism. 1990 May;39(5):525-30. 

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4. 
Soeters MR, Soeters PB, Schooneman MG, Houten SM, Romijn JA. Adaptive 
reciprocity of lipid and glucose metabolism in human short-term starvation. Am J Physiol 
Endocrinol Metab. 2012 Dec 15;303(12):E1397-407. 
5. 
Samra JS, Clark ML, Humphreys SM, Macdonald IA, Frayn KN. Regulation of lipid 
metabolism in adipose tissue during early starvation. Am J Physiol. 1996 Sep;271(3 Pt 
1):E541-6. 
6. 
Horowitz JF, Coppack SW, Klein S. Whole-body and adipose tissue glucose 
metabolism in response to short-term fasting in lean and obese women. Am J Clin Nutr. 
2001 Mar;73(3):517-22. 
7. 
Gjedsted J, Gormsen LC, Nielsen S, Schmitz O, Djurhuus CB, Keiding S, et al. 
Effects of a 3-day fast on regional lipid and glucose metabolism in human skeletal muscle 
and adipose tissue. Acta Physiol (Oxf). 2007 Nov;191(3):205-16. 
8. 
Mansell PI, Macdonald IA. The effect of starvation on insulin-induced glucose 
disposal and thermogenesis in humans. Metabolism. 1990 May;39(5):502-10. 
9. 
Corvilain B, Abramowicz M, Féry F, Schoutens A, Verlinden M, Balasse E, et al. 
Effect of short-term starvation on gastric emptying in humans: relationship to oral glucose 
tolerance. Am J Physiol. 1995 Oct;269(4 Pt 1):G512-7. 
10. 
Horton TJ, Hill JO. Prolonged fasting significantly changes nutrient oxidation and 
glucose tolerance after a normal mixed meal. J Appl Physiol (1985). 2001 Jan;90(1):155-63.