Close-up of the expressive face of a white mouse

Intermittent Fasting in Mice

Research paper

Intermittent fasting increases energy expenditure and promotes adipose tissue browning in mice
Nutrition 2019 Oct; 66:38-43. doi: 10.1016/j.nut.2019.03.015. 2019
Bo Liu , Amanda J Page , Amy T Hutchison , Gary A Wittert , Leonie K Heilbronn

Associated Institutions

Centre for Nutrition and Gastrointestinal Disease, Adelaide Medical School, University of Adelaide, South Australia and Nutrition and Metabolism Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia

Animal Ethics Committee

This study was approved by the animal ethics committees of the South Australian Health and Medical Research Institute (SAHMRI) and the University of Adelaide.


Adipocyte – a cell specialized for the storage of fat, found in connective tissue.
Adipose tissue – brown fat (as opposed to white fat), storing energy in a smaller space than white fat.
WAT – White adipose tissue (fat) often associated with development of obesity and type 2 diabetes and is essential for energy homeostasis by storing excess energy and releasing lipids in response to energy deficits.
Tissue browning –browning describes the emergence of beige adipocytes in white adipose tissue—a reversible process that represents adaptation to increased exercise. Brown fat is triggered to burn calories when we eat and when we are cold (whereas normal fat stores caleries)
Body weight – a person’s weight measured in kilograms
Energy expenditure – the amount of energy that a person needs to carry out physical functions such as breathing, circulating blood, digesting food, or exercising. Measured in calories.
Fat mass – That portion of the human body that is composed strictly of fat
Uncoupling protein 1 (UCP1) – is a mitochondrial carrier protein found in brown adipose tissue
Messenger RNA (mRNA) is a single-stranded RNA molecule that is complementary to one of the DNA strands of a gene.
Glucagon-like peptide 1 (GLP-1) is a gut incretin hormone inducing post-prandial insulin secretion
Visceral adipose tissue (VAT) – intra-abdominal fat that is located inside the abdominal cavity
Subcutaneous adipose tissue (SAT) – fat below the skin


Intermittent fasting (IF) is a widely practiced eating pattern whereby a person intermittently fasts for a certain number of hours each day and eats during a specific time period. This cycle of eating and fasting prolongs the period when the body has burned through the calories consumed during the last meal and begins burning fat. It does not refer to which foods to eat, but rather when foods should be eaten.

Hypothesizing that IF may reduce energy expenditure by stimulating white adipose tissue browning, the ‘researchers’ subjected lean and diet-induced obese mice to an intermittent fasting regime for a period of 8 weeks. The project also studied IF in overweight or obese females.

The mice experiments

24 ten-week old C57BL/6J male mice (from the Bioresources, SAHMRI) were housed four per cage at the Bioresources facility under a 12:12h light/dark cycle with lights on at 7 am and temperature at 21 ±30C. For a period of 8 weeks they were fed a chow diet or a lard based high-fat diet prior to randomizing mice onto either ad libitum feeding (8 mice) group or a group who were required to undergo intermittent fasting for an additional 8 weeks (3 non-consecutive days per week) (16 mice). At 28 weeks of age, all mice were killed, and their inguinal and gonadal adipose tissue were collected.
Food intake, energy expenditure, and inguinal (groin) and gonadal (testis) fat pads were collected in fed or fasted conditions. Subcutaneous adipose tissue was also collected at the start of the experiment and after 8 weeks of IF (in the fed state, and after a 24-hour fast).

The human study

This research also included a clinical trial (approved by the Royal Adelaide Hospital and the University of Adelaide) of intermittent fasting in overweight women. Fifty healthy women who were overweight and obese, aged 35-70 years were randomly assigned to one of two IF groups for 8 weeks. Participants were provided with ~30% of their daily energy requirements for breakfast and then initiated a 24 hour fast on 3 non-consecutive days per week. On fed days, one group was provided foods at ~100% of energy requirements, to achieve an overall 30% energy deficit (IF70). The other group was provided foods at ~145% of their daily energy requirements on fed days without overall energy restriction (IF100). Biopsies were taken at baseline, after 8 weeks of intervention diet and after a 12 hour overnight fast and 24 hour fast. In total 22 participants in each group completed the intervention. Statistical analysis was undertaken.


The publication noted that intermittent fasting reduced body weight and energy intake in high fat diet fed mice and reduced gonadal and inguinal fat pad weights in both groups of mice. It confirmed previous evidence that had already suggested that IF induces browning of white adipose tissue in mice which could be a key mechanism contributing to a healthier phenotype and that IF increases energy expenditure (1,2). It also referred to numerous previous studies confirming that chow fed IF mice compensate for food deprivation by overeating on re-feeding days and do not lose weight.

The results of the experiment also noted the difference in results between mice and humans in that intermittent fasting increased energy expenditure and promoted white adipose tissue browning in mice yet did not alter UCP1 mRNA levels in subcutaneous adipose tissue in women. The researchers also note that only women were studied in the trial and thereby speculating that gender rather than species could explain discrepancies in results.

Relevance to Humans

Humane Research Australia questions why, if a human-relevant component of the study could be undertaken, did the ‘researchers’ attempt to study the effects in a completely different species under unnatural laboratory conditions?

The aims and outcomes of the mouse and human studies are markedly different and it is difficult to ascertain from the study design how the ‘researchers’ planned to draw appropriate and relevant parallels between the two cohorts (animals and humans), and instead it appears that, the mice were used ‘for interest’ without practical applications. In fact the results discuss the different outcomes in mice as compared to the results in the human clinical participants. Surely this indicates that the results from the mice studies cannot be reliably translated to the human condition?

Limitations of using animals to model humans in this type of experiment include the lack of biological variability, lack of co-morbidities or other human risk factors and also the physiological impacts related to stress, thereby distorting the results.

For example, mice have a different caloric requirement to humans and have a dissimilar gastrointestinal pathway and metabolise food much faster. They also absorb and distribute food differently to humans.

Fasting in mice for periods of 24 hours should not be seen as equivalent to a 24 hour fast in humans. Mice have a much faster basal metabolism compared to humans, with a gastrointestinal transit time of 6 hours (3) compared to 40 – 70 hours in humans.(4) Mice also tend to be grazing feeders adapted to a more constant food intake opposed to the ‘meal’ intake of humans (this is highlighted in Figure 1C of the paper with meals frequently 25+ per day in test mice).

Further, metabolic outcomes are influenced by temperature. The temperature of the laboratory in which the animals are housed will therefore impact on the results. According to Weldon, et al, (2015)(5) the gut microbiota of laboratory mice has been studied extensively, noting that gut microbiota profoundly affects the biology of the animal and noting that the environments of different research institutions can also affect the mouse microbiota.

An added complication is that the researchers noted that chow fed IF mice compensate for intermittent food deprivation by overeating on refeeding days and acknowledge that greater increases in food-induced thermogenesis may have partially contributed to increased energy expenditure.

Animal Welfare Concerns

The mice in this experiment were housed in an artificial environment which would have in itself been stressful. Further HRA is advised that a 24 hour fast would have much more marked physiological effects and would elicit more stress in a mouse model compared to a human model. The paper does not make any mention of monitoring for stress behaviours in the mouse subjects. The paper makes mention of some mice dragging food away: while this could be a ‘hunger’ correlated behaviour, hoarding food is also a common mouse behaviour which has been denied to all fasting test subjects.

Cost Benefit Assessment

University resources required to provide laboratory space, the purchase of animals and provide appropriate maintenance and handling by staff, government funding, and not least, animal lives, have been wasted on this experiment, the key finding of which is not applicable to humans.

With human IF studies conducted internationally, including the research institutions involved, HRA questions why animal studies were determined necessary.


The most reliable research methods for understanding intermittent fasting in humans are those that study humans. There is much available data available in the scientific literature on studies involving human subjects.

For example, a very recent systematic review on the effectiveness of IF with potentiate weight loss or muscle gain in humans younger than 60 years old (Sandoral et al) (6) was published this year (2021). In undertaking the review, multiple databases (up to May 2020) were searched with 10 original articles evaluated. The Review noted IF could be beneficial in resistance trained subjects and in overweight individuals to improve body composition by decreasing fat mass and at least maintaining muscle mass, decreasing GOP-1 levels (that regulate appetite after a meal) and improving health-related biomarkers such as glucose and insulin levels.

Further, another review in 2020, Lee, et al (7) noted that although both mouse and human studies present positive metabolic effects on IF, they pointed out “Although food intake and housing environment (temperature and circadian rhythms) are tightly controlled in mouse studies, these are highly variant in clinical research. Particularly, temperature has a substantial impact on browning of WAT under IF intervention.

Human clinical trials

There are many clinical studies relating to intermittent fasting providing human relevant data and illustrating the exiting scientific understanding of IF in humans including the following.

Details and results of a human clinical study in relation to alternate day (IF) undertaken by the University of Graz, Switzerland can readily be found here : Alternate Day Fasting Improves Physiological and Molecular Markers of Aging in Healthy, Non-obese Humans – PubMed (

A search of indicates that there are approximately 90 human intermittent fasting trials around the world relating to the impact on a variety of medical conditions.

And recently (2021) the ABC’s Catalyst program The Truth About Fasting – Catalyst ( followed an experiment designed by Dr Joanna McMillan which took a group of 5 Australians through a fasting experiment over a period of 6 weeks to observe the effect on their bodies. The participants were divided into groups of 5 hours fasting/2 hours eating or 16 hours fasting /8 hours eating. It involved no calorie counting, no medications but just nutritious food. Participants were from different ethnic backgrounds, different sexes, different occupations, adopted different diets to each other and different family histories of illness. All these different factors which can only be observed in the human and which this animal study was unable to provide. Dr McMillan tracked the participants noting symptoms as they presented – again symptoms that animal studies would not be elucidate. One participant noted headaches and nausea and another mood swings. Further, it was also noted that benefits came from diet improvement, again not a factor that could be studied in animals.


Funding for this experiment came from various government sources. The human study was funded by a National Health and Medical Research Council Project Grant APP1023401. Heilbronn was supported by an Australian Research Council Future Fellowship Number FT120100027 ($877,396.00). Liu was supported by an Australian Government Research Training Program Scholarship.

What you can Do

Please use the form below to tell the University of Adelaide and the South Australian Health and Medical Research Institute how disappointed you are with these experiments which subject mice to intermittent fasting. Your message will be sent via email to Professor Anton Middelberg, Deputy Vice Chancellor and Vice President (Research) and Prof S Wesselingh, Executive Director of SAHMRI and the Animal Ethics Committee via Research Services at the University of Adelaide.

Or you may compose your own email and personalise your message, ensuring that it is polite. Their respective email addresses are:

Animal Ethics Committee –

and SAHMRI Executive Director, Prof S Wesselingh c/o Executive Assistant –

Some points you might mention are:
• Male 10 week-old mice are not suitable models for attempting to replicate intermittent fasting in humans.
• Mice have a different caloric requirement to humans and have a dissimilar gastrointenstinal pathway and metabolise food much faster than humans, absorbing and distributing food differently.
• Fasting in mice for periods of 24 hours is not the same as a 24 hour fast in humans.
• Results from the mice studies cannot be reliably translated to the human condition.
• The research paper makes no mention of monitoring for stress behaviours in the mice.
• Stress and physiological impacts on the mice have the ability to distort the results.
• The research included a clinical trial of human participants. Why, if a human-relevant component of the study could be undertaken, was an attempt made to study the effects in a completely different species under unnatural laboratory conditions?

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1. Li, G., et al (2017) Intermittent fasting promotes white adipose browning and decreases obesity by shaping the gut microbiota Cell Metab 26(5) p.801
2. Kim, K.H., et al (2017) Intermittent fasting promoted adipose thermogenesis and metabolic homeostasis via VEGF-mediated alternative activation of macrophage Cell Res 27(11) ; 1309-1326
3. Padmanabhan, P. et al (2013) Gastrointestinal transit measurements in mice with 99mTc-DTPA-labeled activated charcoal using NanoSPECT-CT EJNMMI Res 3:60
4. Gastrointestinal Transit
5. Weldon, L et al (2015) The Gut Microbiota of Wild Mice PLoS ONE 10 (8)
6. Sandoval, C., et al (2021) Effectiveness of intermittent fasting to potentiate weight loss or muscle gains in humans younger than 60 years old: a systematic review Int J Food Sci Nutri 72(6): 734-745
7. Lee, J.L., et al (2020) Intermittent Fasting: Physiological implications and outcomes in mice and men Physiology 35; 185-195


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