Case studies - animal-free research

How the Paleo Diet is related to Gut Health (Microbiome) and Heart Health

Article: Long term Paleolithic diet is associated with lower resistant starch intake, different gut microbiota composition and increased serum TMAO concentrations

Authors: Angela Genoni 1 , Claus T Christophersen 2 3 , Johnny Lo 4 , Megan Coghlan 3 5 ,
Mary C Boyce 4 , Anthony R Bird 6 , Philippa Lyons-Wall 2 , Amanda Devine 2

1School of Medical and Health Sciences, Edith Cowan University, 270 Joondalup Drive,
Joondalup, WA, 6027, Australia.
2School of Medical and Health Sciences, Edith Cowan University, 270 Joondalup Drive,
Joondalup, WA, 6027, Australia.
3School of Molecular and Life Sciences, Curtin University, Bentley, WA, 6102, Australia.
4School of Science, Edith Cowan University, Joondalup, WA, 6027, Australia.
5Forensic Biology, PathWest Laboratory Medicine, Nedlands, WA, 6009, Australia.
6CSIRO Health and Biosecurity, Gate 13, Kintore Avenue, Adelaide, SA, 5000, Australia.

Sources of Support:
The first author was the recipient of a postgraduate research scholarship from Edith Cowan
University and Commonwealth Scientific and Industrial Research Organisation (CSIRO).


The Paleolithic (Paleo) diet is a popular food regime said to boost gut health. The diet includes
whole foods such as fruit, vegetables, seeds and meat and excludes processed goods such as
dairy, grains and soft drinks. Some believe this dietary pattern is better for human health because
the digestive system has not adjusted to more modern food processes.
The goal of this research is to understand if there is a relationship between the Paleo diet and gut
and heart health.


Paleolithic diet: Based on the eating patterns of Hunter Gathers that involves the consumption
of whole unprocessed foods such as fruit, vegetables, meat, fish and seeds and excludes dairy,
grains and legumes.
Gut health: A term used to describe the composition of bacteria that live in the gut
Resistant starch (RS): A carbohydrate that is not digested but fermented by gut bacteria to make
short-chain fatty acids (SCFA).
Trimethylamine-N -oxide (TMAO): Produced by some gut bacteria often from meat, fish and
eggs and linked to an increased risk of heart diseases like atherosclerosis (plaque in the arteries),
stroke and heart attack.
Whole grains: An unrefined grain that contains all three parts of the seed: endosperm, germ, and


Resistant starch (RS) levels, gut bacteria, short chain fatty acids (SCFA) and TMAO metabolite
were measured using a range of techniques as described below. Dietary intake, blood cholesterol
and fat levels were also recorded. These features were measured to show possible gut/heart
health effects from the Paleo diet compared to a standard diet.
Participants were men and women aged 18-70 years old with BMI <30kg/m2 and were nonsmokers

44 were long time followers of the Paleo diet and these were split into two groups: 22
Strict (those who consumed < 1 serve of grains and dairy a day) and 22 Pseudo (> 1 serve of
grains or dairy a day). 47 Controls consumed a diet that included foods from a variety of food
groups including dairy, legumes and grains or alternatives.
Participants completed 3-day weighted diet records to keep track of the quantity and type of
food they were eating. This record included 2 week days and 1 weekend day. After this time,
blood, urine and stool samples were also collected from participants and analysed.
The 3-day weighted diet records were examined by a nutritionist with Australian software
‘FoodWorks’ (v8.0) that gave insight into the types of foods eaten.

Resistant Starch (RS)
Foods included in 3-day weighted diet records were also assigned RS values based on
information from NZFoodFiles 2014 and other reputable sources. This score indicated how
much RS participants ate.
Short Chain Fatty Acids (SCFA)
The SCFAs are a by-product from bacteria breaking down RS in the intestine. These were
measured using Gas Chromatography (GC) and Mass Spectrometry (MS) of stool samples.
Samples were prepared then heated to a gas with GC to separate the individual components.
These gases pass through a column of unreactive gas to the Mass Spectrometer. Here, molecules
are charged and separated based on their mass-to-charge ratio. The results display as a mass
spectrum graph which is used to determine SCFA based on its known pattern or mass.
TMAO was calculated from participant blood serum using Liquid Chromatography (LC) and
tandem Mass Spectrometry (MS-MS). LC involves separating the solution into its components
by pushing it through a column within a moving (liquid) phase under high pressure. After it has
been pushed through, this fluid gets sent to MS-MS where particles undergo further changes of
ionisation, breaking and separating. This process helps form products to detect TMAO via the
mass spectrum displayed.
Gut Bacteria
Stool samples were used to investigate the types of bacteria in participants, using Illumina MiSeq
to sequence the 16S rRNA gene in the V4 region of bacteria. This technology includes several
steps such as breaking up DNA into pieces, creating clusters of these DNA pieces, and adding
coloured tags that show up on camera. There is a different colour for each DNA base and the
order of colours can be matched to a genetic sequence and linked to a known genetic profile for
a bacterium.


RS intake was lower in Strict Paleo and Pseudo Paleo groups compared to controls. In a similar
way, the TMA-making bacteria genus Hungatella was present at higher levels in both Paleo groups
compared to controls. Also, other “good” gut bacteria Roseburia and Bifidobacterium were lower in
Paleo groups compared to controls.
In addition to this, TMAO levels was higher in the Strict Paleo group only compared to PseudoPaleo and controls.

Higher TMAO/Hungatella further correlated with lower levels of wholegrains in the diet.
There were no significant differences in SCFA between all groups. However, Paleo groups
reported a significantly greater intake of fat and the strict Paleo group recorded higher


This study demonstrates a clear link between the Paleo diet and markers of gut/heart health.
Lower RS in Paleo groups is in line with a reduction or lack of grain and legume products.
Similarly, reduced wholegrains may help explain higher Hungatella, a bacteria genus that makes
TMA, a precursor for TMAO. Furthermore, increasing wholegrains in the diet may be important
in keeping Hungatella levels down and improving the balance of gut bacteria.
A big reduction in Roseburia and Bifidobacterium in Paleo groups gives more evidence that the diet
can change the presence of gut bacteria. These two groups of bacteria are known to be beneficial
in gut health and preventing inflammatory diseases – so lower levels could be harmful.
The higher fat in Paleo diets may also justify why there were no changes observed to SCFA. The
higher fat consumption and cholesterol in this diet, in addition to higher TMAO, may increase
the risk for heart disease in the future.
With all things considered, this research indicates the Paleo diet may not be protective for gut
and heart health.


The Paleo diet is a popular diet; however, its claims of positive gut health have needed more
scientific research to back them up. Studies like this suggests that the Paleo diet may actually
cause more harm than good to gut and heart health. These findings are important as they allow
consumers to make more informed decisions about their diets.
This study taps into an exciting area of new research: the gut microbiome. The gut microbiome
is all of the bacteria that live inside your gut. Some of these bacteria are good and others more
harmful in larger quantities. Research like this suggests diet plays a big role in the balance
between good and bad bacteria and connected health problem.

HRA comment

Early research that involves the gut microbiome has shown it is very complex and may be
controlled by a range of factors including genetic background, lifestyle conditions and
behavioural choices. In addition to this, gut bacteria in humans often fails to colonise in nonhuman models. 

Hence, it may be difficult to research the human gut microbiome in an animal setting, so humans are the most ideal subjects



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