TYPE Original Research

PUBLISHED 23 August 2022

DOI 10.3389/fmicb.2022.922727

Intermittent fasting positively

OPEN ACCESS modulates human gut microbial
EDITED BY
Benoit Chassaing,
Institut National de la Santé et de la
diversity and ameliorates blood
Recherche Médicale (INSERM), France

REVIEWED BY
lipid profile
Ndeye Coumba Ndiaye,
INSERM UMRS1256
Nutrition-Génétique et Exposition aux Muhammad Nadeem Khan1 , Sidra Irshad Khan1 ,
Risques Environnementaux (NGERE), Madeeha Ilyas Rana1 , Arshad Ayyaz2 ,
France
Mindy Engevik, Muhammad Yousaf Khan3 and Muhammad Imran1*
Medical University of South Carolina,
1
United States Department of Microbiology, Faculty of Biological Sciences, Quaid-I-Azam University, Islamabad,
Pakistan, 2 Department of Biological Sciences, Faculty of Science, University of Calgary, Calgary, AB,
*CORRESPONDENCE
Canada, 3 Department of Pathology, Pakistan Institute of Medical Sciences (PIMS), Islamabad,
Muhammad Imran
Pakistan
mmimran@qau.edu.pk

SPECIALTY SECTION
This article was submitted to
Microorganisms in Vertebrate
Digestive Systems, Aim: The aim was to evaluate the impact of intermittent fasting (IF) on
a section of the journal human body mass index (BMI) and serum lipid profile thorough constructive
Frontiers in Microbiology
rectification of gut microbiota.
RECEIVED 18April 2022
ACCEPTED 13 July 2022 Methods and results: Fourteen healthy women and thirty-one men were
PUBLISHED 23 August 2022
included in the study. Their blood and fecal samples were collected before
CITATION
Khan MN, Khan SI, Rana MI, Ayyaz A,
and at the end of the study. Blood parameters, anthropometric values,
Khan MY and Imran M (2022) and gut microbiology were noted to investigate the impact of intermittent
Intermittent fasting positively
fasting (IF) on human gut microbiota and physiology. Our data revealed
modulates human gut microbial
diversity and ameliorates blood lipid that IF reduces the body weight and improves blood lipid profile, such as
profile. increasing high-density lipoprotein (HDL) and decreasing total cholesterol,
Front. Microbiol. 13:922727.
doi: 10.3389/fmicb.2022.922727 triglycerides, and low- and very low-density lipoprotein levels. IF also
COPYRIGHT
decreases culturable aerobic bacterial count and increased fungal count. It
© 2022 Khan, Khan, Rana, Ayyaz, Khan was also found that the gut metagenome is altered considerably after IF.
and Imran. This is an open-access
The human fecal bacterial diversity exhibited significant changes in decreased
article distributed under the terms of
the Creative Commons Attribution overall bacterial population, increased bacterial diversity (alpha diversity),
License (CC BY). The use, distribution and promoted evenness within the bacterial population at the species level.
or reproduction in other forums is
permitted, provided the original Anti-inflammatory bacteria Lactobacillus and Bifidobacterium were favorably
author(s) and the copyright owner(s) increased, while pathogenic bacteria were decreased.
are credited and that the original
publication in this journal is cited, in Conclusion: Collectively, these results indicated that IF could improve lipid
accordance with accepted academic
practice. No use, distribution or profile and body weight in humans, and the potential mechanisms might be
reproduction is permitted which does via regulating gut microbiota.
not comply with these terms.
Significance and impact of the study: We demonstrated for the first time
that IF improved body weight and blood lipid profile, indicating that IF could
mitigate gut microbiota in humans.

KEYWORDS

human gut microbiota, intermittent fasting, serum lipid profile, BMI, obesity

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Khan et al. 10.3389/fmicb.2022.922727

Introduction foods and fluids aiming to restrict food intake for a defined
period of time is an easy alternate, is simple to follow, and has
Metabolic disorders and associated diseases like diabetes, shown equal effectiveness in insulin sensitivity, cardiovascular
cardiovascular diseases, inflammatory bowel disease, and functions, weight loss, and other health biomarkers (Rizza et al.,
various cancers including breast and colorectal cancer are the 2014; Mattson et al., 2017).
leading causes of death worldwide (Gibson and Macfarlane, Here, before and after controlled experimental study in
1995; Berg, 1996; Arumugam et al., 2011; Faulds and humans was designed to test the effect of intermittent fasting
Dahlman-Wright, 2012; Rajilić-Stojanović et al., 2015; Blake on weight loss, body mass index (BMI), blood lipid profile,
and Suchodolski, 2016). The diversity and composition C-reactive protein (CRP), and gut microorganisms of obese,
of gut microbiota have been identified as an important normal, and lean but otherwise healthy adult volunteers. We
contributor to metabolic associated disorders, as healthy demonstrate that IF improves BMI and reduces triglycerides
individuals contain highly diverse gut microbiota, while and low-density lipoprotein levels. Intriguingly, our results also
diseased individuals harbor aberrant and less diverse microbiota demonstrate that the IF not only decreases gut microbe numbers
in the gut (Friedman, 2009; Le Chatelier et al., 2013; Harmsen but also increases their diversity and promotes the growth of
and de Goffau, 2016). Moreover, the use of antibiotics microbial species that increase bodily energy metabolism, as
in infants predisposes them to unusual weight gain and identified by the metagenomic analyses.
deposition of adipose tissues later in life, reaffirming that
disruptions in gut microbiota composition can lead to the
onset of obesity and then metabolic diseases (Cox and Blaser, Materials and methods
2015; Turta and Rautava, 2016). Fecal transplantation from
healthy individuals has been proposed to restore normal This study was carried out at the Department of
microbiota. Several studies have demonstrated that fecal Microbiology, Faculty of Biological Sciences, Quaid-i-Azam
transplantation from healthy individuals improved metabolic University, Islamabad, Pakistan.
disorders (cardiovascular disease, type diabetes, and obesity) in
diseased individuals (Elinav et al., 2019). Although effective,
this approach can lead to a reshuffling of gut microbiome Study population and setup
dysbiosis and the emergence of pathobionts (Glauser, 2011;
Weil and Hohmann, 2015). Similarly, other available weight The Intermittent Fasting 91 (16/8 IF) or lean gain method
loss strategies and modulation of gut microbiota by using was employed in this study as previously described (Berkhan,
antibiotics, prebiotics, and probiotics have many limitations. 2010). According to this method, participants abstain from
Like antibiotics kill good microbiota along with pathogens, foods and liquids for at least 16 h for 26 days. The remaining
similarly, it is challenging for probiotic strains to persist in 8 h of the day serve as the feeding window. During this time,
the gut for a longer period. The impact of prebiotic products users may eat as many (or few) meals as desired, with the most
is also not specific (Druart et al., 2014; Erejuwa et al., frequent iteration being two and three meals.
2014). Gonzalez’s group has recently demonstrated in mice
that intermittent calorie restriction activates brown adipose
tissue and ameliorates obesity by most likely modulating the Approval of the study
composition of gut microbiota (Li et al., 2017). In humans,
fasting reduces LDL, Vldl, and triglyceride levels, as well The study was approved by the Bioethical Committee
as increases HDL levels, as observed in type 2 diabetes (BEC) of Quaid-i-Azam University, Islamabad, Pakistan, under
patients (Shehab et al., 2012). Intermittent fasting also enhances protocol number BEC-FBS-QAU2018-109, and all the methods
cardiovascular health, regulates blood pressure by reducing were performed in accordance with the relevant guidelines
brain natriuretic peptide levels, enhances renal activity (Masoro, and regulations advised by the Bioethical Committee (BEC) of
2005; Fontana et al., 2010; Abargouei et al., 2012; Trabelsi et al., Quaid-i-Azam University, Islamabad, Pakistan.
2012; Tiboura et al., 2015), and therefore has become a popular A campaign for the person-to-person meeting was
practice around the globe to achieve effective weight loss and conducted to meet the students (both male and female). The
improve metabolism. It is still not known how intermittent protocol and expected outcomes of the study were orally
fasting influences the gut microbiota in humans and whether the explained to everyone during the meetings. Students who
regime can support the growth of microbial communities that agreed to participate were enrolled in the study. The age of the
increase metabolism associated with increased thermogenesis. volunteers ranged from 18 to 35 years.
Also, continuous energy restriction requires a well-defined diet Informed consent for study: An informed consent was
plan that has met with non-compliance issues and adaption. obtained from all the participants for participation in the
Intermitted fasting or complete abstention from all or selected study, handling of the samples, and data anonymously, and

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confidentially was also ensured by the research team through following the protocols (Wu et al., 2010; Escobar et al., 2014).
this consent (sample of the consent is given in Supplementary Soon after collection, each fecal sample was separated into two
material in Supplementary Figure 1). Information about diet portions. One portion was stored at −80◦ C for later use and
and living habits was recorded for all volunteers (given in DNA extraction. In total, 1 g of the fecal sample was taken,
the Supplementary material in Supplementary Figure 2). diluted in sterile phosphate buffer saline (PBS), taken this as
Participants were asked to follow the protocol. They were asked initial dilution, and diluted serially. In total, 100 µl from each
to continue their ad libitum eating habits during eating widows dilution was poured and spread on different culture media for
and live as per routine. the isolation of fecal microorganisms (Castillo et al., 2006).
The volunteers who followed the protocol completed the For total microbial aerobic count (TMAC), BHIA (Brain Heart
study and provided the required samples (two blood samples Infusion Agar; Oxoid United Kingdom) media were used. The
and two fecal samples), and their data were included in the de Man–Rogosa–Sharpe Agar (MRSA, Oxoid United Kingdom)
current manuscript for the conclusion of the results. The with 5.4 pH was used to selectively grow the Lactobacillus
volunteers who were unable to follow the protocol, or complete spp. The M17 (Oxoid United Kingdom) was used to grow
the study, or were not able to provide the required samples Enterococcus, Streptococcus, and Lactococcus spp. MacConkey
were excluded from the conclusion of the results. Statistical agar was used to grow the family of Enterobacteriaceae, and
analysis (mean, standard deviation, standard error, paired t-test, OGA (Oxytetracycline Glucose Agar; Oxoid United Kingdom)
and agglomerative hierarchical clustering) was performed using was used for fungal growth. The inoculated plates were
Microsoft Excel 2016 and XLSTAT 2019.1.2. incubated at 37◦ C for 48–72 h (Lim et al., 2004). After
Initially, an equal number of male and female volunteers incubation, plates were examined for microbial growth. The
were selected for the study, but there was a dropout in color, size, and morphology of the grown colonies were noted.
the samples, and finally, 31 male volunteers and 14 female The total number of colonies on each plate was counted using a
volunteers were able to complete the study and provide colony counter. An average of 30–300 colonies was used for the
the samples. Furthermore, volunteers were placed in any actual colony count.
group of lean, underweight, normal, and overweight or obese
where applicable.
Selection criteria included healthy (physically and mentally Extraction of metagenomic DNA from
fit upon looking and feeling), non-smokers, no antimicrobial use stool
(in last 4 weeks), no laxatives use (in last 2 months), no weight
reducer use, no diarrhea (in last 1 week), no gastrointestinal tract Favor prep Stool DNA isolation mini kit (Favorgen) was
disease, no exercise more than 10 h/week, and no supplement used for DNA extraction. Following their designed protocol,
consumption; those participants provided the samples (two 100mg stool was taken in a microtube to which 200mg glass
blood samples and two fecal samples), followed the protocol, beads were added; frequently, 300 µl of SDE1 buffer and 20 µl
and completed the study. of Proteinase K were transferred to the same microtube for
disruption of microbial cells. Then, this mixture was vortexed
at high speed for 5–7 min and was incubated at 70◦ C first for
Blood collection and analysis 10 min and for an additional 5 min to lyse gram-positive cells.
To enhance homogenization, the stool sample was vortexed
In total, 10 ml of blood (at once) was collected from the three times during the period of incubation. The sample was
volunteers two times in specified blood collection tubes. One let to cool and added 100 µl of SDE2 buffer. The sample
sample was collected 2 days earlier to the study, and the second was kept on an ice pack for 5 min and then centrifuged for
was collected on the 25th or 26th day of IF (upon convivence). 5 min at 14,000 rpm. The supernatant was taken and shifted
The samples were processed soon after collection, and serum to another microtube with the addition of SDE3, 200 µl buffer.
was separated and kept at −20◦ C until further analysis. Different The mixture after mixing was incubated for 2 min at room
parameters were recorded from blood analysis. These factors temperature. The mixture again was centrifuged at said speed
include C-reactive proteins, serum total cholesterol level, serum for 2 min, and the supernatant was transferred to another sterile
triglycerides level (TG), high-density lipoproteins (HDL) levels, microtube. In total, 1 µl of RNase of concentration 100 mg/ml
low-density lipoproteins (LDL) levels, and very low-density was transferred to the supernatant for the degradation of RNA.
lipoproteins (vLDL) levels. After removing the drops from the lid via spinning, it was
supplemented with 250 µl SDE4 buffer and 250 µl cooled
ethanol and mixed by pulse overtaxing. Collection columns
Fecal sample collection and analysis were positioned in the collection tube, and the sample was
shifted to it. The sample was centrifuged at maximum speed;
The stool samples were collected twice from volunteers. the column was shifted to another collection tube, and flow-
The samples were collected in sterile stool collection containers through was discarded; columns were transferred to the next

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clean collection tube, and flow-through was wasted. Wash buffer single run. It performs de novo assembly and generates contigs
(750 µl) was used for washing the impurities by adding them (FASTA) without using a reference genome. It also generates
to the column and then centrifuging at maximum speed for FASTQ files that can be used by third-party tools for analysis.
2 min. This step was repeated. For drying the column and It further aligns the reads against the reference genome and
avoiding residual contamination, the collection column was generates a sample report (bam format). Finally, it classifies
centrifuged for 3 min. bacteria according to their 16SrDNA.
The elution tube was taken, and the column was shifted to
it. The 70 µl elution buffer was transferred right to the center
of the column for elution of the DNA. For complete absorbance Analysis of the data
of elution buffer into the column, it was centrifuged later to the
addition of elution buffer. DNA was eluted after centrifugation All physiochemical data was analysed for statistical
and was stored at −20◦ C for further processing. significances by using R software (version 3.3.3). One way
and two-way ANOVA was used, and results are presented as
highly significant (p < 0.01) and significant (p < 0.05). The
Processing of extracted metagenomic post hoc test; Student Newman Keuls used to differentiate means
DNA for sequencing after ANOVA significance. In addition to this the two-sample
Wilcoxon signed-rank test was used to compare serum lipid
Amplification and purification of extracted DNA profile before and after IF.
PCR was carried out for the amplification of the V4 Raw sequencing data analysis was done by using MRDNA
region of the 16S rRNA gene using forward primer 515F pipelines (MR DNA, Shallowater, TX, United States). Errors
(GTGCCAGCMGCCGCGGTAA) and reverse primer 806R were minimized, and noises were removed. Finally, operational
(GGATCACNVGGGTWTCTAAT). The amplification was taxonomic units (OTUs) were generated, and chimeras were
carried out in triplicates using a volume reaction of 20 µl extruded. OTUs were further defined by clustering at 3%
(Caporaso et al., 2011). HotStar Taq Plus Master Mix (Qiagen, divergence (similarity 97%). Final OTUs were classified
Valencia, CA, United States) and samples were used for the taxonomically using BLASTn against a database derived from
preparation of reaction volume. PCR was performed at these GreenGenes, RDPII, and RDPI (Desantis et al., 2006). Diversity
settings: early denaturing, 94◦ C for 3 min, then tailed by 30 analysis was done by using QIME2 Microbiome.
cycles of denaturation at 94◦ C for 30 sec, annealing at 53◦ C
for 40 s, and finally amplification at 72◦ C for 1 min. After
the entire process, confirmation of the amplification and
Results
quality of the gene fragment were tested in 2% agarose gel.
Then, as per molecular weight and concentration of DNA, all
the samples were joined to each other and were purified by
Impact of intermittent fasting on BMI
using calibrated Ampure XP beads (Bioscience Corporation,
The impact of IF on weight was personalized, the weight
MA, United States).
in the majority of normal-weighted male volunteers (25/28;
p = 0.040) did not change, and only three volunteers reduced
their weight, but their BMI remained in the normal range (18.5–
Library preparation
24.9; p = 1). The weight of underweighted male volunteers
(2/3) increased (1–2 kg) (p = 0.040). The weight of normal
Following the Illumina TruSeq DNA library preparation
female volunteers (5/16) increased significantly (p = 0.001)
protocol, libraries were prepared from the input of 1 ug of total
within the normal BMI range (18.5–24.9), while underweighted
DNA along with the aforementioned primers via the Illumina
female volunteers (2/2) body weight increased and the weight
TruSeq PCR-Free Library Preparation Kit.
of obese/overweight (3/3 out of total 14) decreased significantly
(p < 0.001) (Figure 1 and Tables 1, 2).

Sequencing

Sequencing was performed at MRDNA sequencing Effect of intermittent fasting on acute

laboratory (Shallowater, TX, United States) using Illumina inflammatory biomarker blood
MiSeq next-generation sequencer. The MiSeq platform C-reactive protein
performs clonal amplification, genomic DNA sequencing, and
data analysis (from assembly to functional classification) with Blood C-reactive protein (CRP) was negative for all subject
base calling, alignment, variant calling, and reporting in a samples (data not shown).

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FIGURE 1
Experimental design of the study and summary of the anthropometric results. (A) Experimental design. (B) Summary of the anthropometric
values [one-way ANOVA was used, highly significant (p < 0.01) and significant (p < 0.05) values are presented with *** and *, respectively, while
non-significant values are presented by N.S].

Effect of Intermittent fasting on serum from 155 to 133 mg/dl (Figure 2B and Supplementary
lipid profile Tables 1, 2), low-density lipoprotein decreased from 104
to 93 mg/dl and from 102 to 87 mg/dl (Figure 2C
By using two-sample Wilcoxon signed-rank test, significant and Supplementary Tables 1, 2), and very low-density
alternation was noticed in total triglycerides (p = 0.032), total lipoprotein decreased from 21 to 19 mg/dl and from 19
cholesterol (p = 0.008), and low-density lipoprotein (p = 0.004) to 18 mg/dl (Figure 2D and Supplementary Tables 1, 2),
except in very low-density lipoprotein (p = 0.117). Total while total high-density lipoprotein (HDL) increased from
triglycerides decreased from 104 to 100 gm/dl and from 89 27 to 28 mg/dl and from 28 to 31 mg/dl (Figure 2E and
to 95 mg/dl (Figure 2A and Supplementary Tables 1, 2), Supplementary Tables 1, 2) for both the female and male
total cholesterol decreased from 152 to 141 mg/dl and samples, respectively.

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Intermittent fasting alters the diversity designed by using the number of reads, OTUs, Shannon,
and composition of the fecal Simpson index, and PCoA (Figure 3 and Table 3). Generally, the
total number of OTUs was high in the samples collected before
microbiota
IF while decreasing in samples collected at the end of the study.
At the phylum level, 99% of the intestinal microbiota
No differences were observed in cultured aerobic bacteria
of this study subject consisted of nine phyla namely
and fungi (Supplementary Figures 1A–E). The impact of
intermittent fasting on gut microbiota by culture-independent Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria,
microbiota was observed. In this assay, ten representative stool Cyanobacteria, Verrucomicrobia, Spirochaetes, Lentisphaerae,
samples from individual groups of the human cohort were and Tenericutesirmicutes. Except for one group (male
profiled through 16S DNA sequencing. Reads obtained from underweight before fasting), Firmicutes were the dominant
sequencing were filtered, low-quality reads were removed, and phylum among all the groups. In the female overweight/obese
the remaining reads were classified on phylum, genera, and group, in response to intermittent fasting, Firmicutes decreased
species level. Similarity tagging was 97% which confirmed from the mean percentage of 60.47 to 54.43, Proteobacteria
the identification of all the species. Richness, α diversity, increased from 4.17 to 22.61%, Bacteroidetes decreased from
beta diversity, and dominance of the bacterial population are 22.57 to 11.57%, and Actinobacteria decreased from 12.12

TABLE 1 Detail of the anthropometric values of the male volunteers.

Sample ID Age Height Weight before Weight at the BMI before BMI at Fold change BMI status
(years) (inches) study (Kg) end of study study end of BMI Before/After
(Kg) study study

M1 24 66 65 65 23.13 23.13 NIL Normal

M2 22 66 60 60 21.35 21.35 NIL Normal
M3 22 64 65 65 24.59 24.59 NIL Normal
M4 23 71 57 57 17.52 17.52 NIL under weight
M5 22 67 57 57 19.68 19.68 NIL Normal
M6 23 69 64 62 20.83 20.18 NIL Normal
M7 22 69 65 64 21.16 20.83 0.95 Normal
M8 23 63 54 54 21.08 21.08 NIL Normal
M9 22 68 70 70 23.46 23.46 NIL Normal
M10 22 71 68 68 20.90 20.90 NIL Normal
M11 23 63 56 56 21.86 21.86 NIL Normal
M12 22 65 54 54 19.80 19.80 NIL Normal
M13 23 64 53 53 20.05 20.05 NIL Normal
M14 23 67 58 58 20.02 20.02 NIL Normal
M15 24 68 66 63 22.12 21.11 0.95 Normal
M16 24 60 75 72 32.28 30.99 0.96 Obese
M17 23 63 50 50 19.52 19.52 NIL under weight
M18 22 68 63 61 21.11 20.44 0.97 Normal
M19 23 65 54 54 19.80 19.80 NIL Normal
M20 24 60 53 53 22.81 22.81 NIL Normal
M21 23 64 57 57 21.56 21.56 NIL Normal
M22 22 77 57 58 14.89 15.16 1.0 under weight
M23 24 64 63 60 23.83 22.70 0.95 Normal
M24 24 68 68 66 22.79 22.12 0.97 Normal
M25 24 68 67 65 22.45 21.78 0.97 Normal
M26 24 65 58 58 21.27 21.27 NIL Normal
M27 24 72 68 68 20.32 20.32 NIL Normal
M28 25 69 70 68 22.78 22.13 0.97 Normal
M29 23 62 53 53 21.36 21.36 NIL Normal
M30 24 72 65 64 19.43 23.12 1.2 Normal
M31 23 66 56 56 21.34 21.34 NIL Normal

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TABLE 2 Detail of the anthropometric values of the female volunteers.

Sample ID Age Height Weight (Kg) Weight (Kg) at BMI before BMI at Fold change BMI status
(years) (inches) before study the end of study study end of BMI
study

F1 25 64 52 52 19.67 19.67 NIL Normal

F2 23 62 62 62 24.99 24.99 NIL Underweight
F3 35 64 94 90 35.56 34.05 0.96 Obese II
F4 30 68 115 110 38.54 36.86 0.96 Obese II
F6 24 63 53 63 20.69 24.60 1.19 Normal
F7 23 62 62 59 24.99 23.78 0.95 Overweight/normal
F9 24 63 52 63 20.30 24.60 1.21
F11 25 64 55 62 20.81 23.45 1.13 Normal
F12 25 65 62 57 22.74 20.90 0.92 Normal
F14 25 62 58 56 23.38 22.57 0.97 Normal
F20 24 64 50 64 18.91 24.21 1.28 Normal
F23 23 63 55 58 21.48 22.64 1.05 Normal
F25 22 62 45 62 18.14 24.99 1.38 Normal
F29 34 63 63 63 24.60 24.60 NIL Underweight
Normal

to 11.26%. In the female normal group, Firmicutes increased changed to 64. Underweight male volunteer’s genera increase
from the mean percentage of 60.47 to 63.81, Proteobacteria from 19 to 52 (Figure 5).
increased from 4.17 to 5.5%, Bacteroidetes decreased from In the female overweight/obese group, Roseburia faecis was
22.57 to 17.41%, and Actinobacteria decreased from 12.12 to the dominant genera making up 15.34% of all the bacterial
6.90%. Similarly, in the female underweight group, Firmicutes population that decreased to 0.97% at the end of the trial. The
decreased from the mean of 86.91 to 84.54%, Proteobacteria Prevotella copri was the second most dominant group with a
increased from 1.57 to 2.95%, Bacteroidetes decreased from 3.86 percentage of 11.57 that decreased to 7.01% during intermittent
to 2.18%, and Actinobacteria increased from 5.53 to 8.92%. fasting. The third most populous genera, Bifidobacterium
The effect of intermittent fasting on male volunteers adolescentis, also decreased from 8.41 to 5.80% at the end of the
is unique for both groups. In the male normal group, study. The Faecalibacterium also decreased from 5.76 to 2.91%
Firmicutes decreased from 69.95 to 40.42% at the end at the end of the study. The Ruminococcaceae, before fasting,
of intermittent fasting, Proteobacteria increased from 0.95 had 4.82% of the overall bacterial population that decreased to
to 24.78%, Bacteroidetes increased from 8.87 to 19.81%, 2.91%. Some genera also increased in number when observed at
and Actinobacteria decreased from 19.72 to 6.43%. In the the end of the study trial, for example, Shigella sonnei to 16.35%
male underweight group in response to intermittent fasting, and Clostridium spp increased to 14.4% at the end of the trial.
Firmicutes increased from the mean percentage of 26.37 to 58.91, The results for other genera are depicted in Figure 6A, and detail
Proteobacteria decreased from 73.28 to 1.56%, Bacteroidetes is given in Supplementary Table 4.
increased from 0.11 to 2.31%, and Actinobacteria increased from In the female normal volunteers’ cohort, the Campylobacter
0.19 to 37.14%. Alpha diversity was not significantly altered, spp. were the dominant one making up 16.94% of the total
while beta diversity was shown significantly altered in obese fecal microbiota population that decreased to 0.12% at the end
(Figure 4). The results for the remaining phyla are given in of the study trial. The Clostridiales also changed from 7.28
Supplementary Table 3. to 1.3% at the end of the trial. The Puniceicoccales initially
The number and type of bacterial genera altered in response was the third major group with 7.23% of the total, but at the
to intermittent fasting. Comparative abundance analysis of end of the study, it was not detected in the countable form.
bacterial diversity involved 365 genera. In total, 82 genera The Bifidobacterium adolescentis also changed in response to
above 0.1% were identified in the fecal microbiota of the intermittent fasting from 5.61 to 0.2%. The Sphingobacteriales
female overweight/obese group before intermittent fasting and initially was 5.28% that later on changed to neglectable count.
57 genera after fasting. In contrast, we found that women Dominant genera at the end of the intermittent fasting in female
of normal BMI had 64 genera before fasting and 83 genera normal volunteers cohort were Ruminococcus, Eubacteriaceae,
after intermittent fasting. It was also observed that underweight Barnesiella, Clostridium, and Subdoligranulum with respective
women had 63 genera before fasting and 61 after intermittent percentages of 12.60, 10.91, 5.44, 5.34, and 4.82. For more
fasting. Normal male volunteers had initially 21 genera that results, see Figure 6B and Supplementary Table 5.

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FIGURE 2
Physiological parameters of blood serum lipid profile of the volunteers before and after intermittent fasting: (A) triglycerides, (B) total
cholesterol, (C) low-density lipoproteins, (D) very low-density lipoproteins, and (E) high-density lipoproteins. Two-sample Wilcoxon
signed-rank test was used, all parameters shown a Significant (p < 0.05) alteration except vLDL (p = 0.117).

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FIGURE 3
Weighted mean relative abundance of bacteria by 16S rDNA region at the phylum level for female and male volunteers before and after
intermittent fasting.

TABLE 3 Detail of the bacterial diversity of all the samples.

Metagenomic Female Male

data
Obese Normal Underweight Normal Underweight

Before After Before After Before After Before After Before After
fasting fasting fasting fasting fasting fasting fasting fasting fasting fasting

No. of Reads 389328 250457 370675 390956 263153 261845 320605 370675 336297 407028
OTUs 197 223 212 215 205 200 194 212 193 206
(Genera)
Genera 82 57 64 83 64 61 21 64 19 52
with ≥ 0.1%

*All the numbers are given in mean.

Before intermittent fasting, the fecal microbiota of fasting, the five dominant genera were Ruminococcus (35.46%),
the female underweighted volunteer’s cohort contained Bifidobacterium (12.31%), Eubacterium (7.42%), Clostridium
Clostridium spp. Being the most prominent genera with (6.33%), and Oscillospira (5.04%). Later after the intermittent
18.01% that the end of the trial increased to 24.23%, fasting trial, the five prominent genera were Campylobacter
with Clostridium disporicum being the second most spp. (16.94%), Clostridiales (728%), Puniceicoccales (7.23%),
prominent genera with 8.63% of total fecal microbiota. Bifidobacterium adolescentis (5.61%), and Sphingobacteriales
The Ruminococcus was the third highest in number (7.23%), (5.28%). Detail for the remaining genera is given in Figure 7D
and the Erysipelotrichaceae was the fourth one (6.79%). At and Supplementary Table 7.
the end of the intermittent fasting trial along with clostridium, Similarly, it was observed that the fecal microbiota of
the Eubacteriaceae with 6.97% and the Bifidobacterium underweighted male volunteers has changed in response to
adolescentis with 3.63% were also among the dominant intermittent fasting. Before the trial, Pseudomonas trivialis
genera. Detail of the other genera is given in Figure 6C and (69.47%), Brevibacillus limnophilus (16.23%), Lysinibacillus
Supplementary Table 6. sphaericus (4.63), and Bacillus (3.64) were dominating while
In the male normal volunteers’ cohort, the number and at end of the trial Bifidobacterium adolescentis (28.12%),
type of genera also changed. It was observed that before Ruminococcaceae (25.68), Lactobacillus ruminis (9.15%),

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FIGURE 4
Alpha and beta diversity in volunteer’s intestinal microbiota in response to intermittent fasting. Diversity is shown for each sample and
comparative for treatment. (A) Alpha diversity (Shannon index), (B) alpha diversity (Simpson index), and (C) beta diversity (PCoA). The box plot
shows diversity for each sample and for experimental group.

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FIGURE 5
Heat tree of the abundance of taxa at different ranks of the 23 tree hollows in New South Wales, Australia. The size and color of nodes and
edges are correlated with the abundance of taxa. The central node is the total of all the other nodes in the tree for each phylum.

Catenibacterium mitsuokai (5.55%), and Clostridiales (3.58%). 65.65% of the total bacterial flora. Initially, there were 24
Detail for the remaining genera is given in Figure 6E and bacterial genera unique, which increased to 55 later on at the
Supplementary Table 8. end of the study (Figure 7E). The number of core bacterial
In the female samples, the overweight/obese group had genera in all the groups before intermittent fasting was 130
167 bacterial genera as core, and it makes 66.0% of the total (46.26%) (Figure 7F) and after intermittent fasting was 147
bacterial count. At the start of the study, this group had (49%) (Figure 7G).
30 genera unique while at the end of the study it had 56
genera unique (Figure 7A). The female normal group had
181 bacterial genera as the core that makes up 73.57% of the Discussion
total bacteria present in the fecal sample. Initially, it had 31
bacterial genera which increased to 34 at the end of the study Human physiology is significantly impacted by trillions
(Figure 7B). The female underweight group had 176 (76.85%) of gut microbiota having nine million non-redundant genes
bacterial genera as the core; at the start of the trial, 29 unique (Hooper and Gordon, 2001; Yang et al., 2009). Dysbiosis of
genera were associated with this group later on reduced to 24 the gut microbiome causes a variety of metabolic disorders
(Figure 7C). In the male samples, the normal group had 174 that leads to chronic inflammations which are depicted by
bacterial genera as a core that makes up almost 75% of the overweight and obesity as phenotypic outcomes (Vuksan
total bacterial flora. In the remaining 25%, initially, it had eight et al., 2016). Body weight is a physical indicator of metabolic
unique genera, while at the end of the study, there was an diseases (Swanton et al., 2007). Different strategies like the
increase in unique species and it had 38 genera “Figure 7D.” use of probiotics (Fooks and Gibson, 2002), prebiotics, fecal
The male underweight group had 151 species as core, making transplantation (Delzenne et al., 2013), and the use of antibiotics

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FIGURE 6
Mean weighted distribution of dominant intestinal bacteria at genus level in response to intermittent fasting: (A) obese female volunteers, (B)
normal female volunteers, (C) underweight female volunteers, (D) male normal volunteers, and (E) male normal volunteers.

are being tried to restore the gut microbiota to normal. In this In this study, it was found that IF has an impact on
context, fasting is considered an ancient therapy (Handschin, body mass indexes, blood lipid profiles, and gut microbial
2016). Numerous research studies find out that IF mostly profiles. It was found that observing IF reduced the BMI of
results in weight loss and improvement of blood profile obese/overweight individuals, and there was a slight reduction
(Aloui et al., 2016). of BMI in normal men, while no change was found in

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FIGURE 7
Venn diagram representing core unique and shared intestinal microbiota of female and male volunteers in response to intermittent fasting.
(A) Obese/overweight female volunteers, (B) normal female volunteers, (C) underweight female volunteers, (D) male normal volunteers, (E)
male underweight volunteers, (F) all volunteers before intermittent fasting, and (G) all volunteers after fasting.

normal women. Surprisingly, weight increase was observed in different nations and reported that people using normal diets
underweight women. These results show that this IF regimen with physical activities have improved lipid profiles after IF
tends to normalize the total body mass without its unwarranted while people eating more or energy-rich diets or doing no or
depletion in healthy individuals. Reduction of body weight by limited physical activities have no change in their lipid profile
IF has been reported (Adlouni et al., 1997; Ley et al., 2006; Kul at the end of study (Abdelgadir et al., 2015). Yeoh et al. (2015)
et al., 2014); however, normalization of body weight is reported performed a study in 2015 on 29 volunteers in Singapore, and at
for the first time. The number of Bifidobacteria and Lactobacillus the end of the study, he concluded that weight was reduced, and
increased in all five groups, and it is well established that reduced lipid profile was also improved in volunteers who were taking
body weight is linked to an increasing trend in gut microbial routine diets and doing physical activities (Yeoh et al., 2015).
diversity, and overweight or obesity is linked to reduced gut It was also observed that the IF trial lowered the level of
bacterial diversity (Ley et al., 2006). In our study, the weight total cholesterol but retained it in the normal recommended
reduction was found linked to an increasing trend in bacterial range (<200 mg/dl). The level of triglycerides decreased in
diversity, while a decreasing trend in bacterial diversity was 24/31 male volunteers while increased in 5/31 male volunteers.
observed especially in women those gained weights. In an earlier In the same way, the level of triglycerides is retained in
report, the impact of IF on a female subject showed an increase the normal range (< 150 mg/dl). The level of low-density
in weight was due to the use of energy-dense diets and less lipoprotein (LDL) decreased in 20/31 male volunteers and
physical activities during the fasting days (Sadeghirad et al., increased in 6/31 male volunteers in the same recommended
2014; Savitri et al., 2016). However, the impact of IF is not ranges of <100 mg/dl. The level of high-density lipoprotein
gender-specific, when tested in the mice model (Piotrowska (HDL) increased in 24/31 and decreased in 3/31 male volunteers
et al., 2016). Abdelgadir in 2015 conducted a study on three but in the normal range (>40 mg/dl), and the level of very

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Khan et al. 10.3389/fmicb.2022.922727

low-density lipoproteins decreased in 22/31 male volunteers had the highest Firmicutes of 69.95% and later decreased to
and increased in 6/31 male volunteers. The total cholesterol 40.42%. The Actinobacteria also reduced from 19.72 to 6.43%,
level decreased in all female volunteers. The total triglycerides while Proteobacteria increased from 0.95 to 24.78%, Bacteroides
decreased in 11/14 female volunteers and increased only in three from 8.87 to 19.81%, and Verrucomirobia from 0.21 to 7.98% at
volunteers, but within the normal recommended range, low- the end of the study. In female underweighted group, Firmicutes
density lipoproteins decreased in 12/14 female volunteers, and and Bacteroides decreased from 63.18 to 38.55% and 17.41 to
HDL increased in 13/14 female volunteers. IF lowered total 5.43%, respectively, while Proteobacteria increased from 5.52
aerobic bacterial count in all the male and female volunteers to 41.96% and Actinobacteria from 6.90 to 13.97% at the end
when checked through conventional culturing methods. The of the study. Female overweight/obese group before IF carried
number of total Lactococcus and total Lactobacillus, when high Firmicutes than Bacteroides and low Proteobacteria. At the
assessed through culturing method, seen increased at the end of end of the study, Firmicutes and Bacteriodes were decreased
the IF trial in both male and female volunteers. and Proteobacteria was increased up to 11%. In female normal
Recently, a study established the link between gut bacterial group, no significant changes at phylum level were observed. It
diversity and lipid blood profile, which showed that fasting has been concluded that too high and too low Proteobacteria are
sugar level, total triglycerides, total cholesterol, and low-density signs of dysbiosis. A minor percentage of Proteobacteria (<12%)
lipoproteins are associated with reduced gut bacterial diversity is known healthy one at phylum level (Shin et al., 2015).
as compared to the control cohort (Rebolledo et al., 2017). At the genera level, before IF, it was seen that the normal-
Another study conducted on Finnish-men also showed that weighted male group had a high percentage of Pseudomonas
elevated blood lipids are associated with increased gut bacterial triavilis, that is, 69%. This bacterium is isolated from grasses
diversity (Org et al., 2017). Yang et al. (2017), in 2017, and is kept in risk group one, and it may cause infection
conducted a study on pigs, which showed that increased in humans (Söhngen et al., 2013). Later, at the end of the
blood lipid levels are associated with altered bacterial diversity study, it decreased to 0.04%. The Brevibacillus limnophilus was
while Lactobacillus is negatively correlated with the blood in the second number (4%) followed by Bacillus spp. At the
lipid profile (Yang et al., 2017; Liu et al., 2018). The in vitro end of the study, the number of Ruminococcus flavefaciens
cholesterol assimilation activity of gut microbiota isolated from increased to 28%, Bifidobacterium adolescentis to 28%, and
healthy individuals has been confirmed by different researchers Lactobacillus rumins to 9%. A study conducted by Cucchi et al.
recently, and the presence of bile salt hydrolase bsh genes (2013) showed that the fecal material of vegetarians has a
is reported in Lactobacillus and Bifidobacteria, mostly used high number of Ruminococcus, Lactobacillus, and Bifidobacteria
for cholesterol assimilation activities (Pereira and Gibson, sp. Participants consumed a vegetable-rich diet during the
2002; Begley et al., 2006; Kim et al., 2009, 2013). These study period. The Ruminococcus flavefaciens are fiber digesting
bacteria are known for anti-inflammatory activities, and inulin bacteria mainly involved in interactive digestion and acquisition
is the key component that favorably increases the number of the nutrients in the intestine (Cucchi et al., 2013). Ejtahed
of Bifidobacteria spp (de Souza et al., 2017). These findings et al. (2012), investigated and found that increase in the number
favored our results where all the samples carried Lactobacillus of Bifidobacterium is responsible for blood cholesterol lowering.
spp while their lipid profile was also positively improved. It In another study the effect of energy restriction on body weight
has been demonstrated that overweight individuals carry a and gut microbiota was evaluated. It was concluded that an
high microbial load in their intestines when compared with increase in the number of Bifidobacterium sp and lactobacillus
normal ones (Mokkala et al., 2016). Another study showed that spp is associated with weight loss (Santacruz et al., 2009).
increased bacterial richness is associated with parasite-positive A normal-weighted male group at the end of the IF
guts (Andersen et al., 2016). It was explained by Maine study had 5% Catenibacterium mitsoukai of the total bacterial
Margo that increased diversity in the intestine is considered population. This species is heterofermentative and has a
a healthy flora. They have more potential to degrade and broad range of substrate utilization, thus providing more
metabolize diverse nutrient components (Maine and Kelly, nutrients to their host (Kageyama and Benno, 2000). Before
2016). Therefore, the reduction in bacterial richness and IF, there were no detectable species of Collinsella aerofaciens,
increase in diversity confirm a positive alteration in the but later on, it was observed that they make up 2% of
intestinal bacteria. Changes were seen in the percentages of all the total bacterial population present in the gut. The
bacterial phylum at the end of the study (Figure 8). Normal Collinsella aerofaciens was associated with the prevention
weight male group had a higher percentage of Proteobacteria and cure of irritable bowel diseases (Kassinen et al., 2007).
73.28% which decreased to 1.56% by intermittent fasting. This group carried 2% Subdoligranulum spp. These species
Firmicutes were initially 26% while increased to 58% making have been used for the treatment of type 2 diabetes (Wang
the highest portion of all the phylum at the end of the study. and Jia, 2016). Less number of Dorea spp was observed,
Increase in Actinobacteria from 0.19 to 37.14% was observed and it is known that an increased number of Dorea spp
at the end of the study. Male underweighted group before IF can contribute to the development of irritable bowel disease

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FIGURE 8
Hierarchically clustered heatmap of the bacterial distribution of different communities. Double hierarchical dendrogram shows the bacterial
distribution. The bacterial phylogenetic tree was calculated using the neighbor-joining method, and the relationship among samples was
determined by Bray–Curtis distance and the complete clustering method. The heatmap plot depicts the relative percentage of each bacterial
phylum (variables clustering on the vertical axis) within each sample group (horizon-axis clustering). The relative values for bacterial phylum are
indicated by the color intensity with the legend indicated at the bottom of the figure.

(Rajilić-Stojanović et al., 2015). These findings present an copri (16%). At the end of IF, they were decreased to 1
improvement in the health status of the intestine and the and 8%, respectively. Increased Roseburia spp. are associated
whole host. In the male underweighted group, evenness and with gall stone formation (Tamanai-Shacoori et al., 2017),
bacterial diversity were increased after the trial. It carried more while increased Prevotella copri is associated with autoimmune
percentage of Campylobacter spp, that is, 16% of the total diseases (Campbell, 2014).
bacterial population. Studies have shown that Campylobacter Strength of the study: Blood lipid profile was improved
spp are normally present in animals and poultry, and their and modulated gut microbiota even though the volunteers were
count goes high in warmer months. Not all Campylobacter taking their ad libitum diets during the eating windows of the
spp are pathogenic to humans (Keener et al., 2004). The experiment and were living their routine life. No dietary or
Campylobacter spp. have shown the colonization of the large living habits were advised to be changed or restricted, but, in
intestine (Saint-Cyr et al., 2016). This study was performed many gut modulation studies, experimental subjects are passed
in warmer months, while the volunteers were consuming through dietary restriction or changing living styles. Recently,
poultry meat as a part of their routine diet. Hence, the high a study conducted on a mice model for the improvement of
concentration of Campylobacter sp in the fecal samples of blood lipid profile and modulation of gut microbiota kept the
the volunteer’s cohort might be the competitive exclusion experimental mice restricted to a diet having the main portion
of these undesired microorganisms by the intestinal flora. of isoquercetin and inulin (Tan et al., 2018). In another study,
The Megasphaera elsdenii were 5% of the total bacterial antibiotics were administered in the diet for the improvement
population. This bacterium is responsible for the treatment of of blood lipid profile and modulation of gut microbiota (Rune
metabolic acidosis (Marx et al., 2011). The Oscillospira spp. et al., 2016). We have conducted our experimental trial on the
were decreased in the groups. According to a study, a decrease human population, and it reflects the actual outcome and can be
in Oscillospira spp is considered anti-obesity signature bacteria used as a reference for further studies, while in many cases, such
(Walters et al., 2014). Before IF, overweight/obese female group studies are performed on mice model. There is a difference in the
carried a high number of Roseburia spp (17%) and Prevotell gut microbiota of humans and mice which affects the outcome of

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any experimental trial. Recently, the gut microbiota of mice and Conflict of interest
humans was compared using large datasets, and it was found
that both the gut microbiota are similar at phylum level but The authors declare that the research was conducted in the
have large variations at the species level (Xiao et al., 2015). The absence of any commercial or financial relationships that could
core microbiota of mice is small, while the core of human gut be construed as a potential conflict of interest.
microbiota is large (Kostic et al., 2013). It was found that 85%
of genera that are present in mice are not present in humans
(Nguyen et al., 2015), while, when compared at the gene level,
only 4% of genes were found in mice microbiota (Xiao et al.,
2015). The gut microbiota of mice can be changed by the diet Publisher’s note
within 1 week, while human gut microbiota does not change so
quickly (Wang and Jia, 2016). The difference between the results All claims expressed in this article are solely those of the
of women and men may be due to the fasting days, and women authors and do not necessarily represent those of their affiliated
were exempted to fast during their menstrual cycle. The number organizations, or those of the publisher, the editors and the
of fasting days is not defined yet. reviewers. Any product that may be evaluated in this article, or
Limitation of the study: The number of male and female claim that may be made by its manufacturer, is not guaranteed
samples was not equal and thus may have an effect on the results. or endorsed by the publisher.
This may have influenced the results.

Data availability statement Supplementary material

The datasets presented in this study can be found in The Supplementary Material for this article can be found
online repositories. The names of the repository/repositories online at: https://www.frontiersin.org/articles/10.3389/fmicb.
and accession number(s) can be found below: https://www.ncbi. 2022.922727/full#supplementary-material
nlm.nih.gov/bioproject/PRJNA477600, PRJNA477600.
SUPPLEMENTARY FIGURE 1
Informed consent form for participants.
SUPPLEMENTARY FIGURE 2
Ethics statement Diet and living habits of Participants.
SUPPLEMENTARY FIGURE 3
The studies involving human participants were Impact of intermittent fasting on culturable aerobic bacteria and fungi
reviewed and approved by BEC-FBS-QAU2018-109. The from fecal material of participants.

patients/participants provided their written informed consent SUPPLEMENTARY TABLE 1

Impact of intermittent fasting of serum lipid profile of male participants.
to participate in this study.
SUPPLEMENTARY TABLE 2
Impact of intermittent fasting of serum lipid profile of female
participants.
Author contributions SUPPLEMENTARY TABLE 3
Impact of intermittent fasting of gut microbiota at phylum level of
participants.
MI and MIR conceived the study and experimental
SUPPLEMENTARY TABLE 4
designing. MI supervised the study. AA and MI analyzed the Impact of intermittent fasting of gut microbiota at genera level of obese
data and critically revised the manuscript. MYK contributed to female participants.
sample analysis. MNK and SIK conducted experimental work SUPPLEMENTARY TABLE 5

and drafted the manuscript. All authors contributed to the Impact of intermittent fasting of gut microbiota at genera level of
normal weight female participants.
preparation of the final manuscript.
SUPPLEMENTARY TABLE 6
Impact of intermittent fasting of gut microbiota at genera level of
under-weight female participants.
Acknowledgments SUPPLEMENTARY TABLE 7
Impact of intermittent fasting of gut microbiota at genera level of
normal weight male participants.
We would like to acknowledge Hafiz Abrab Sikandar
SUPPLEMENTARY TABLE 8
for his guidance and all the volunteers of the study who Impact of intermittent fasting of gut microbiota at genera level of
participated willingly. under-weight male participants.

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