Table 2 Characteristics of included interventional studies

1

Kong et al. 2014 [43]

Clinical trial

59

France

45 overweight and obese subjects

(6 M, 39 F);

(mean BMI: 33.2±0.55)

14 healthy females as reference group

Mean BMI: 22.62

Three clusters with 7-day dietary records; Cluster 1 with the least healthy

eating behavior (n=14), Cluster 3 the healthiest eating behavior (n=13), and Cluster 2 was in-between clusters 1 and 3 in terms of healthfulness (n=18)

qPCR, Metagenomic sequencing

no significant difference across the clusters (p>0.05)

↓ hsCRP and IP10

↑ HAM56+cells

the healthiest dietary cluster had the highest microbial gene richness

↓ confectionary and sugary drinks,

↑ Fruits, yogurts and soups

NA

NA

NA

2

Haro et al. 2015 [44]

Prospective randomized controlled trial

20

Spain

20 male patients with obesity

Mean age: 63.3 y

Mean BMI: 32.2

Two randomized groups first receiving Med diet (35% fat, 22% monounsaturated) and second receiving

LFHCC diet (28% fat, 12% monounsaturated) for one year

16S rRNA sequencing

↓Prevotella (p=0.028)

↑Roseburia (p=0.002)

↑Oscillospira (p=0.016)

↑Parabacteroides

↑P. distasonis (p=0.025)

Protective effects on the development of type two diabetes

by increasing in the insulin sensitivity measured by OGTT

Alpha diversity via Chao1/ no significant correlation

Beta diversity via UniFrac/ no significant correlation

NA

NA

Changes in 7 out of 572 metabolites in feces and 3 out of 697 metabolites

in plasma were related to the changes in bacterial species

NA

3

Haro et al. 2016 [37]

Prospective controlled trial

239

Spain

239 patients with CHD with last coronary event over last six months in two groups;

138 metabolic syndrome patients,

101 healthy individuals

two healthy diets: a

MD and a LFHCC, for two years in the gut microbiota of MetS patients and those in the control group

16S rRNA sequencing

↑Parabacteroides

↑Bacteroides

↑Faecalibacterium

↑Bifidobacterium

↑P. distasonis

↑B. thetaiotaomicron

↑F. prausnitzii

↑B. adolescentis

↑B. longum

(p-values<0.05)

NA

NA

NA

NA

NA

14-item food questionnaire

4

Haro et al. 2017 [38]

Prospective randomized controlled trial

106

Spain

106 subjects with CHD with last coronary event over last six months in three groups;

33 obese men with severe metabolic disease;

32 obese men without metabolic diseases;

41 non-obese men

differences in bacterial community at baseline and after 2 years of dietary intervention following consumption of two healthy diets; MD and low-fat

16S rRNA sequencing

Bacteroides↑

Prevotella↑

Faecalibacterium↑

Roseburia↑

Ruminococcus↑

(p-values<0.05)

P. distasonis↑ (p=0.014)

F. prausnitzii↑ (p=0.043)

NA

Alpha diversity via Chao1 and Faith’s PD/ no significant correlation

Beta diversity via UniFrac/ no significant correlation

↑vegetables,

↑ fruit,

↑ cereals,

↑potatoes, ↑legumes,

↑ dairy products,

NA

NA

14-item questionnaire

5

Djuric et al. 2017 [41]

Randomized dietary intervention trial

93

USA

Healthy individuals at increased risk of colon cancer as defined;

Participants were randomized to MD / Healthy Eating diet;

Biopsy data was available from 88 participants at baseline and 82 participants after six months

16S rRNA sequencing

No significant changes in colonic mucosal bacterial community (p>0.05)

NA

Alpha diversity via Shannon and inverse Simpson/ no significant correlation

Beta diversity via community distance index / no significant distinction between control and case group. Only significant within healthy arm after 6 months dietary intervention

30% of calories from fat as polyunsaturated: saturated: monounsaturated fatty acids (PUFA: SFA: MUFA) ratio of 1:2:5.

NA

NA

NA

6

Luisi et al. 2019 [45]

Randomized controlled trial

18

Italy

Healthy controls

(6M, 12 F)

Mean BMI: 21.6,

Mean age: 41.4

Overweight individuals (11M, 7 F)

Mean BMI: 30.152

Mean age 52.1 y

18 overweight/obese subjects (BMI ≥25) and 18 normal weight controls (BMI 18.5–24.9) were fed with MD enriched for three months. Feces and blood samples were collected at baseline and after three months

qPCR for rRNA-polymerase β subunit

↑ Lactic Acid Bacteria (p<0.05)

↓ Inflammatory cytokines

↓ Oxidative stress

↓ Myeloperoxidase

↓ 8-hydroxy-2-deoxyguanosine

↑ IL-10

NA

NA

NA

NA

• Specific score from 0 to 18 for adherence

7

Pagliai et al. 2019 [46]

A crossover study

23

Italy

over- weight individuals (16 F, 7 M)

Mean age: 58.6 ± 9.8 y

healthy subjects were randomly assigned to isocaloric MD or VD diets lasting 3-months each and then crossed

16S rRNA sequencing

↑Lachnoclostridium (p=0.039)

↑Enterorhabdus (p=0.003)

↑Parabacteroides (p=0.037)

↑Clostridium sensu stricto (p=0.005)

↑Veillonella (p=0.029)

↓Anaerostipes (p=0.048)

↓ Inflammatory cytokines:

↓VEGF,

↓MCP-1,

↓IL-17,

↓IP-10

↓IL-12,

Alpha diversity via Simpson and Shannon/ no significant correlation

Beta diversity via UniFrac and Bray-Curtis/ no significant distinction

↑fruit,

↑vegetables, ↑cereals, ↑legumes,

↑olive oil

\(\to\) fish

\(\to\) poultry

\(\to\) dairy products

↓ red meat

↓ wine

GC-MS

↑ propionic acid (p=0.034);

But no effect on butyrate, acetate, isobutyrate, isovalerate or valerate

NA

8

Ghosh et al. 2020 [47]

Randomized, multicenter, single-blind,

612

UK, France, Netherlands, Italy, Poland

non-frail or pre-frail elder subjects

Gut microbiota before and after the administration of a 12 month long MedDiet intervention tailored to elderly subjects

16S rRNA sequencing

↑Faecalibacterium

↑Roseburia

↑Eubacterium

↑Bacteroides

↑Prevotella

↓Anaerostipes

↓Ruminococcus

↓Collinsella

↓Coprococcus

↓Dorea

↓Clostridium

↓Veillonella

↓Flavonifractor

↓Actinomyces

↑F. prausnitzii,

↑R. hominis

↑E. rectale

↑E. eligens

↑E. xylanophilum

↑B. thetaiotaomicron,

↑P.copri

↑A.hadrus

↓R. torques

↓C. aerofaciens

↓C. comes

↓D. formicigenerans

↓C. ramosum

↓V. dispar

↓F. plautii

↓A. lingnae

(p-values were not mentioned)

Lower frailty; Improved cognitive function; ↓Inflammatory markers;

↓CRP

↓IL-17

Alpha diversity via undetermined index/ no significant correlation

↑Fruits

↑Vegetables

↑Wholegrains

↑Legumes

↑Fish

↓Fats

↓Alcohol

↓Sugar

NA

↑ SCFA

↓ Secondary bile acids,

↓ p-cresols,

↓ ethanol

↓ carbon dioxide

(p-values not mentioned)

NA

9

Pisanu et al. 2020 [48]

Randomized controlled ntervention study

69

Italy

Case group: 23 obese/overweight patients with BMI > 25 and being “diet-free” as defined.

(20 F, 3 M)

Mean age: 53±9 years

Control group: 46 individuals normal weight being “diet-free” as defined.

(40 F, 6 M)

Mean age: 49±11 years

The Gut Microbiota of Obese and overweight patients was compared before (T0) and after 3 months (T3) of nutritional intervention by MD

16S rRNA sequencing

↑ Bacteroidetes

↑ Proteobacteria

↓ Firmicutes

↑ Sphingobacteriaceae

↑ Sphingobacterium

↑ Bacteroides

↑ Prevotella stercorea

↑ Proteobacteria

↓ Lachnospiraceae

↓ Ruminococcaceae

↓ Ruminococcus

↓ Veillonellaceae

↓ Catenibacterium

↓Megamonas

↓Sutterella

(p-values<0.05)

Body weight ↓

Fat mass ↓

Alpha diversity via Shannon/ no significant correlation

Beta diversity via Bray-Curtis/ no significant distinction between control and case group after intervention. Only significant at baseline between case and control.

↑Vegetables

↑fruit

↑cereals

↑fish

↑pulses

NA

NA

• MDS (0 to 55)

10

Zhu et al. 2020 [49]

Pilot study

10

USA

Healthy subjects 18-25 years old,

Mean age: 22.1 y

Mean BMI: 24.39

Fast food diet for 4 days followed by Mediterranean diet for 4 days, with a 4-day washout in between

16S rRNA sequencing

↓ Collinsella (p=0.028)

↑ Butyricicoccus (p=0.019)

NA

Alpha diversity via undetermined assessor/ no significant correlation

Beta diversity via UniFrac/ no significant distinction

NA

LC-MS

↑Beneficial metabolites:

↑ indole-3-lactic acid (p=0.003)

↑indole-3-propionic acid (p<0.001)

NA

11

Galié et al. 2021 [34]

Randomized controlled intervention

50

Spain

Metabolic Syndrome patients without T2DM/ any other PMH/DH

Mean age: 51.37 y

(25-60)

Mean weight: 85.1

BMI: 25-35

adults with Metabolic Syndrome were randomized to a controlled, crossover 2-months dietary-intervention trial with a 1-month wash-out period, following a MedDiet or consuming nuts

16S rRNA sequencing

Lachnospiraceae↑ (p<0.05)

Ruminococcaceae↑ (p<0.05)

Glucose↓

Insulin↓

HOMA-IR↓

Alpha diversity via Phyloseq/ no significant correlation

Beta diversity via Bray-Curtis/ no significant distinction

↑Vegetables

↑fruit

↑cereals

↑fish

↑Olive oils

↓Red meat

↓Butter

↓ Sugary beverages

LC-MS

↑ homocitrulline

↑ byacetate,

↑ cadaverine

↑ malate (P values<0.05)

17 items MDS

12

Galié et al. 2021 [35]

Crossover randomized clinical trial

44

Spain

Metabolic Syndrome patients without T2DM/ any other PMH/DH

Age range: 37-65.

BMI: 25-35

crossover 2-months dietary-intervention trial with a 1-month wash-out period, consuming a

MD or a non-MD plus nuts. Nutritional data were collected at the beginning and the end of each intervention period using 3-day dietary records

16S rRNA sequencing

No significant changes in the characteristics and composition of gut microbiota (p>0.05).

Glucose↓

Insulin↓

HOMA-IR↓

NA

↑Vegetables

↑fruit

↑cereals

↑fish

↑Olive oils

↓Red meat

↓Butter

↓ Sugary beverages

GC-MS

LC-MS

↑ HpEPE,

↑ testosterone,

↑ PC

↑ TMA,

↑ succinic acid,

↑ ChoE

↑ taurolithocholic acid,

↑ amino acids

↑ LPC

↑ carnitine species

↑ TG

↑ LPE

(p-values<0.05)

NA

• 17-item MDS

13

Ismael et al. 2021 [39]

Single-arm pilot study

9

Portugal

9 patients with type 2 diabetes (3F and 6M) Age range: 47-77 years

Mean age: 66 ± 9 years

Mean BMI of 27.60 ± 4.03 Kg/m²

12-week single-arm pilot study, participants received individual nutritional

counseling sessions;

indices were assessed at baseline, 4 weeks, and 12 weeks after the intervention

16S rRNA sequencing

Prevotella to Bacteroides ratio↑ (p=0.438)

Firmicutes to Bacteroidetes ratio↑ (p=0.846)

Total Gut bacteria↑

↑ Glycemic control

↓ HbA1c

↓ HOMA-IR

Alpha diversity via Chao1 and Shannon/ no significant correlation

Beta diversity via Bray-Curtis/ no significant distinction

Vegetables

fruits;

grains

cereals;

fish

Legumes

NA

NA

• MEDAS score

14

Muralidharan 2021 [50]

Randomized controlled trial

400

Spain

Overweight/obese subjects Age range: 55–75 years

BMI: 27-40

200 in IG

200 in CG

IG: intensive weight loss lifestyle intervention based on an energy- restricted, MD and physical activity

CG: non-energy- restricted MD For one year

16S rRNA sequencing

Firmicutes ↑↓ (p=NA)

Lachnospiraceae↑ (p-value<0.001)

Butyricicoccus ↓

Haemophilus ↓

Ruminiclostridium ↓

Eubacterium hallii ↓

(p-values<0.05)

↓ BMI

↓ HbA1C

↓ FBS

↑ HDL

Alpha diversity via Chao1 and Shannon/ no significant correlation

Beta diversity via Bray-Curtis and UniFrac/ no significant distinction

NA

NA

NA

• 17-item MDS

15

Rejeski et al. 2021 [51]

pilot study of controlled diets

10

USA

Healthy individual without PMH/DH;

4F, 6M

Mean age:31.8 y

Mean BMI:22.9

Subjects gave a stool sample at baseline and then was provided with prepared meals of a “typical” American diet; after 2 weeks, a second stool sample was collected. All subjects were then provided with prepared meals based on the MD

for another 2 weeks, followed by a final stool sample collection.

16S rDNA sequencing

↑ Akkermansia,

↑ Lactococcus,

↑ Lachnospira

Ratio of Firmicutes/Bacteroidetes ↑

↓ Coprococcus

(p-values<0.05)

NA

Alpha diversity via Simpson/ significantly increased

Beta diversity via Bray-Curtis/ no further association.

Fruits

Vegetables

NA

NA

NA

16

Barber 2021 [52]

cross-over, randomised study

18

Spain

Healthy individual without Gastrointestinal PMH;

Age range: 18–38

BMI range: 19.2–25.5

Each diet (Western-type diet and fibre-enriched MD) was administered for 2 weeks preceded by a 2-week washout diet

DNA quantification

↑ Anaerostipes hadrus

↑ Agathobaculum butyriciproducens

(p-values<0.05)

↑ Gas Evacuation number and volume

↑ Bowel Movement

Alpha diversity via Simpson, Shannon, Chao1, inverse Simpson/ no significant correlation

Beta diversity via Bray-Curtis/ significant association

↑fruits,

↑vegetables ↑legumes

LC-MS

↑ deoxycholate

Glucuronide

↑ 5-hydroxyindole

↑ L-aspartyl-L-phenylalanine

↑ TMAO

(p-values<0.05)

NA

17

Choo et al. 2023 [42]

cross-over, randomised study

34

Australia

age between 45 and 75 years; Adults with SBP \(\ge\) 120mmHg and risk factors for cardiovascular disease; not on hypertensive medication

Patients were randomly assigned to a MD or low-fat control diet for 8 weeks. patients underwent an 8-week washout period

16S rRNA sequencing

↑Butyricicoccus

↑Lachnospiraceae

↑Streptococcus

↓Colinsella

↓Veillonella

(p-values<0.05)

↓ SBP

↑FBS

Alpha diversity via Faith/ no significant correlation

↑fruits,

↑vegetables ↑legumes

↑cereals

NA

NA

Beta diversity via Weighted Unifrac/ no significant association

10-point and 18-point MDS

18

Boughanema et al. 2023 [36]

Single arm trial

91

Spain

91 Patients with obesity and metabolic syndrome;

BMI≥ 27 and ≤40 kg/ m2

Patients were stratified as Low or optimal vitamin D groups on baseline. Both received a hypocaloric MD regimen for one year.

16S rRNA sequencing

↑Bacteroidetes

↑Firmicutes

↑Proteobacteria

(p=0.002 for all three phyla)

↓ Wight

↓BMI (in optimal vitamin D group)

↓HbA1C (in optimal vitamin D group)

↑HDL (only in low vitamin D group)

Alpha diversity via Faith-PD and Chao1/ significant correlation

NA

NA

↑butanoate

(p=0.018)

Beta diversity via Weighted and Unweighted Unifrac/ significant distinction

NA

19

Gomez-Perez et al. 2023 [40]

Single arm trial

297

Spain

NAFLD or NASH cases;

Men aged 55-75 and women aged 60-75;

BMI≥ 27 and ≤40 kg/ m2; patients with a history of CVD or chronic condition were excluded.

participants were stratified into three groups according to alterations in the Hepatic Steatosis Index (HSI) or the Fibrosis−4 score (FIB−4) between baseline and after one year of intervention by MD

16S rRNA sequencing

↑Alcaligenaceae

↑Bifidobacteriaceae

↓Proteobacteria

↓Lentisphaerae

↓Enterobacteriaceae

↑Bifidobacterium

↑Faecalibacterium

↑Sutterella

↑Desulfovibro

↑Lachnospira

↑Oscillospira

↓Blautia

(p-values<0.05)

↓HbA1C

Alpha diversity via Faith-PD and Shannon/ No significant correlation

NA

NA

NA

Beta diversity via Weighted Unifrac/ significant distinction

NA

20

Shoer et al. 2023 [53]

Single blinded randomized control trial

200

Israel

Age range: 18-65

Exclusion criteria:

• Use of diabetes

medications,

• Use of antibiotics three months before enrollment

• Chronic diseases, or chronic use of medications that affect glucose/energy metabolism or HbA1c

200 participants were randomly assigned to a ratio of 1:1 to MD and PPT regimens for 6 months then followed for another 6 months; Participants met two of four glycemic criteria.

DNA quantification

↑Ruminococcaceae

↑Clostridiaceae

↓Eubacteriaceae

↑F. prausnitzii

↓Eubacterium ventriosum

(p-values<0.05)

↑ Glycemic control

↑ Lipid control

Alpha diversity via Shannon index/significant correlation after MD intervention

↑whole-wheat bread and grains

↑legumes

↑fruits ↑vegetables,

↑olive oil

↑fish

↑poultry

↑low-fat dairy products

LC-MS

27 metabolites significantly increased (p<0.05) and no metabolites significantly decreased:

• 10 uncharacterized biochemical

• 7 lipids

• 6 amino acids,

• xenobiotic (3-bromo-5-chloro-2,6-dihydroxybenzoic acid)

• peptide (HWESASXX),

• nucleotide (dihydroorotate)

• bilirubin

NA

NA