Effect of Intensity and Duration of Exercise on Gut Microbiota in Humans: A Systematic Review

Citation: Bonomini-Gnutzmann, R.; Plaza-Díaz, J.; Jorquera-Aguilera, C.; Rodríguez-Rodríguez, A.; Rodríguez-Rodríguez, F. Effect of Intensity and Duration of Exercise on Gut Microbiota in Humans: A Systematic Review. Int. J. Environ. Res. Public Health 2022,19, 9518. https://doi.org/10.3390/ijerph 19159518 Academic Editor: Paul B. Tchounwou Received: 15 June 2022 Accepted: 25 July 2022 Published: 3 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Environmental Research and Public Health Review Effect of Intensity and Duration of Exercise on Gut Microbiota in Humans: A Systematic Review Romina Bonomini-Gnutzmann 1, Julio Plaza-Díaz 2,3,4,* , Carlos Jorquera-Aguilera 1,*, Andrés Rodríguez-Rodríguez 5and Fernando Rodríguez-Rodríguez 6 1Escuela de Nutrición y Dietética, Facultad de Ciencias, Universidad Mayor, Santiago 8580745, Chile; [email protected] 2Children’s Hospital of Eastern Ontario Research Institute, Ottawa, ON K1H 8L1, Canada 3Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, 18071 Granada, Spain 4Instituto de Investigación Biosanitaria IBS.GRANADA, Complejo Hospitalario Universitario de Granada, 18014 Granada, Spain 5Gastric Cancer Research Group—Laboratory of Oncology, UC Center for Investigational Oncology (CITO), Pontificia Universidad Católica de Chile, Santiago 8331150, Chile; [email protected] 6IRyS Group, Physical Education School, Pontificia Universidad Católica de Valparaíso, Valparaíso 2374631, Chile; [email protected] *Correspondence: [email protected] (J.P.-D.); [email protected] (C.J.-A.); Tel.: +34-958241599 (J.P.-D.); +569-95791706 (C.J.-A.) Abstract: (1) Background: The gut microbiota might play a part in affecting athletic performance and is of considerable importance to athletes. The aim of this study was to search the recent knowledge of the protagonist played by high-intensity and high-duration aerobic exercise on gut microbiota composition in athletes and how these effects could provide disadvantages in sports performance. (2) Methods: This systematic review follows the PRISMA guidelines. An exhaustive bibliographic search in Web of Science, PubMed, and Scopus was conducted considering the articles published in the last 5 years. The selected articles were categorized according to the type of study. The risk of bias was assessed using the Joanna Briggs Institute’s Critical Appraisal Tool for Systematic Reviews. (3) Results: Thirteen studies had negative effects of aerobic exercise on intestinal microbiota such as an upsurge in I-FABP, intestinal distress, and changes in the gut microbiota, such as an increase in Prevotella, intestinal permeability and zonulin. In contrast, seven studies observed positive effects of endurance exercise, including an increase in the level of bacteria such as increased microbial diversity and increased intestinal metabolites. (4) Conclusions: A large part of the studies found reported adverse effects on the intestinal microbiota when performing endurance exercises. In studies carried out on athletes, more negative effects on the microbiota were found than in those carried out on non-athletic subjects. Keywords: aerobic exercise; adults; elite athletes; large intestine; gut microbiota 1. Introduction The intestinal or gut microbiota is “the set of microbes that colonize our digestive tract that interact with each other and with the host” [1–3]. Currently, more than one thousand different microbial species have been found that can reside in the human gastrointestinal tract [ 4 ]. Approximately one hundred sixty species are found in the large intestine [ 3 ], developing a biomass of more than 1.5 kg [ 5 ]. The microbiota contain bacteria, as well as fungi, viruses, and protists [ 1 ]. The most abundant and diverse families of bacteria in the adult gastrointestinal tract are Actinobacteria,Bacteroidetes,Firmicutes, and Proteobacteria, and less diverse are Verrucomicrobia,Lentisphaerae,Sinergistetes,Planctomycetes,Tenericutes, and Deinococcus-Thermus [2,3]. Int. J. Environ. Res. Public Health 2022,19, 9518. https://doi.org/10.3390/ijerph19159518 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2022,19, 9518 2 of 17 The composition of the gut microbiota is formed throughout early childhood, induced by genetic and environmental factors [ 1 – 3 ]. The maturation of the intestinal microbiota in the adult type is gone at the age of three years [ 1 , 6 ]. Factors such as age, lifestyle, diet, and genetics can alter the gut microbiota, creating a dynamic ecosystem [ 7 ]. Other related factors could include mode of delivery, geography, breastfeeding, weaning, and exposure to environmental bacteria [ 1 ]. Some researchers proposed that the intestinal microbiota can act as an endocrine organ [ 8 ] and can have an enormous impact on human health, including the immune function, physiology, metabolism, and nutrition of the host [ 9 ]. In the same line, the gut microbiota performs a series of protective, structural, and metabolic functions essential to the health of the host, including food handling, the ingestion of complex polysaccharides not digestible by the host, the movement of pathogens, and the synthesis of vitamins among others [ 10 ]. Observational studies have found that intestinal microbiota may contribute either to the pathogenesis of various common metabolic disorders including type 2 diabetes, obesity, cardio-metabolic diseases, malnutrition, and non-alcoholic liver disease, as well as to the metabolic health of the human host [ 11 ]. Healthy gut microbiota shows an essential role in the configuration of the local and systemic immune function of intestinal bacteria throughout life, favoring the maintenance of tolerance toward antigens of the commensals and activation against antigens of commensal pathogens [ 12 ]. The intestinal microbiota plays an important role in the regulation of host energy metabolism, hydration status, systemic inflammatory responses, and oxidative stress [13]. Physical exercise is described as the implementation of some activity in order to improve or preserve overall health and physical fitness [ 14 , 15 ]. Currently, physical exercise is recognized as a formidable preventive and treatment mediation that is recognized to be efficient in causing benefits for immune and metabolic health [ 15 , 16 ]. Endurance exercise is described as cardiovascular activity, for example: cycling, running, swimming, skiing, and rowing that is performed for a long time, four to six hours per day, six days per week [ 17 ]. This intense exercise includes processes that involve physiological, affective, cognitive–behavioral, and biochemical responses in an effort to recover homeostasis [9]. Some professional athletes experience immunosuppression or gastrointestinal symptoms, such as abdominal pain, diarrhea, or leaky gut syndrome [ 8 ]. Alterations in the intestinal microbiota produced by strenuous exercise can also produce exercise-induced gastrointestinal disorders [ 18 ]. Some symptoms that are stated during the performance of endurance exercise include bloating, nausea, cramps, and diarrhea [ 19 ]. It has been studied that exercising to exhaustion can disrupt the balance among the gut microbiota and the immune system [ 12 ]. Exercise-induced gastrointestinal damage or inflammation could adversely affect athletic routine and, in some cases, have competition dropout [ 20 ]. Another study mentions that intense exercise creates increased gastrointestinal damage, mild endotoxemia, and intestinal permeability [21]. The main finding in post-exercise gastrointestinal problems is possible ischemia–reperfusion injury developing from a momentary interruption of splanchnic blood flow. When the intense physical exercise finishes, we observed triggering reactive oxygen species (ROS) production, damage to the gastrointestinal mucosa, and inflammation [22]. Likewise, it is little known thus far how high-intensity exercise influences the intestinal microbiota [ 21 ]. The importance of knowing the mechanisms in which the intestinal microbiota might have an important role in affecting athletic routine is of significant attention to athletes working to expand their competitive performance and diminish recuperation time during training [ 12 ]. Such information could have an advantage in the comprehension of gut microbiota influences on athlete health [12]. Therefore, the main aim of this systematic review is to elucidate the knowledge of the function played by high-intensity and high-duration aerobic exercise on gut microbiota composition in athletes and how these effects could provide disadvantages in their sports performance. Int. J. Environ. Res. Public Health 2022,19, 9518 3 of 17 2. Materials and Methods 2.1. Search Strategy This systematic review follows the PRISMA guidelines [ 23 ]. An exhaustive bibliographic search of three databases (Web of Science, PubMed, and Scopus) was conducted considering the articles published in the last 7 years (from 1 January 2015 to 31 August 2021). Table 1shows the search strategy in the Web of Science, PubMed, and Scopus databases. This systematic review was listed on the PROSPERO (International prospective register of systematic reviews) website on 5 May 2022, with the following record CRD42022323300. Available from: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD420 22323300 (accessed on 14 June 2022). Table 1. Search strategy in databases. Database Search Strategy Limits Filters Web of Science (ALL (Physical activity AND gut microbiota OR Physical activity AND intestinal barrier OR Physical activity AND intestinal permeability OR Physical exercise AND gut microbiota OR Physical exercise AND intestinal barrier OR Physical exercise AND intestinal permeability)) Title Articles English 238 items filtered PubMed (Physical activity OR physical exercise) AND (gut microbiota OR intestinal barrier OR intestinal permeability) Title Articles English Humans 104 items filtered Scopus TITLE-ABS-KEY (physical AND activity AND gut AND microbiota) OR (physical AND activity AND intestinal AND barrier) OR (physical AND activity AND intestinal AND permeability) OR (physical AND exercise AND gut AND microbiota) OR (physical AND exercise AND intestinal AND barrier) OR (physical AND exercise AND intestinal AND permeability) AND (LIMIT-TO (OA, “all”)) AND (LIMIT-TO (PUBYEAR, 2022) OR LIMIT-TO (PUBYEAR, 2021) OR LIMIT-TO (PUBYEAR, 2020) OR LIMIT-TO (PUBYEAR, 2019) OR LIMIT-TO (PUBYEAR, 2018) OR LIMIT-TO (PUBYEAR, 2017) OR LIMIT-TO (PUBYEAR, 2016) OR LIMIT-TO (PUBYEAR, 2015)) AND (LIMIT-TO (DOCTYPE, “ar”)) AND (LIMIT-TO (LANGUAGE, “English”)) Title Articles English 5934 items filtered 2.2. Selection and Exclusion Criteria The selection criteria were: (i) articles written in English, (ii) databases aforementioned, (iii) human studies, (iv) original articles: clinical trials, randomized controlled trials (RCTs) quasi-experimental, long-term, prospective, and cross-sectional studies, (v) articles from January 2015 to August 2021. The exclusion criteria were: (i) studies that include people with pathologies, (ii) studies that include animals, (iii) studies that comprise children under 18 years of age and older adults (+65 years), (iv) studies that intervened with supplements or some diet, (v) case studies, case reports, letters to the editor, systematic review and meta-analyses and narrative review. No restrictions were placed on the body composition of the trained subject. After removing repeated documents, suitability was measured by evaluation of the manuscript title and abstract and later evaluation of the full text. 2.3. Data Extraction and Reliability The search was carried out by five independent reviewers (R.B.-G., F.R.-R., J.P.-D., C.J.-A. and A.R.-R.). They read the titles and abstracts of all retrieved articles. A meeting was held to resolve disagreements about eligibility. The following information was collected from each included study: the first author, year of publication, type of study, objective, the number of subjects, body mass index (BMI), maximum oxygen consumption (VO 2max ), Int. J. Environ. Res. Public Health 2022,19, 9518 4 of 17 gender, and age when it was available, type of exercise, how the exercise was carried out, the molecular analysis used for the detection of the gut microbiota, the main results obtained, and conclusions. The selected articles were categorized according to the type of study (low-, medium-, or high-intensity or long-term exercise interventions). The results of the studies that met the selection criteria for their recovery were examined. 2.4. Assessment of the Quality and Level of Evidence The risk of bias was assessed using the Joanna Briggs Institute’s Critical Appraisal Tool for Systematic Reviews. In summary, this tool includes four specific checklists depending on the study design (i.e., cross-sectional, quasi-experimental, cohort, RCTs studies). The answers for each of them had four possible categories: “yes” (criterion met) and “no” (criterion not met). Specific tools included: eight items for cross-sectional studies, nine items for quasi-experimental, and thirteen items for RCTs. According to the above, the studies were considered as “low quality” evidence when ≤ 49% of the items were classified as “yes” (criterion met). Following, the articles were considered as “medium quality” evidence between 50–74% of the items were scored as “yes” and “high quality” evidence when ≥ 75% of the items were classified as “yes”. The answers “not applicable” and “nonclear” were excluded by percentage [ 24 – 26 ]. The five reviewers assessed the studies’ quality separately. A consensus meeting was organized to resolve possible differences between the reviewers. 3. Results Figure 1displays the chosen reporting elements for systematic reviews and the flow chart for the search strategy. A total of 6277 studies were located in the three databases assessed. Then, 95 studies were excluded for duplicates and 6136 studies were excluded after reading the title and abstract that were outside the topic of the review. A total of 45 studies were assessed for eligibility. After analyzing the exclusion criteria, sixteen studies were included. Seven studies had an observational design (i.e., five cross-sectional and two long-term designs), and nine studies had an experimental design (eight were quasi-experimental, and one was RCT). Table 2shows the summary of the studies included. This review is focused on data from 513 participants, and the sample size ranged from 4 to 86 subjects. Two of the sixteen studies involved only women [ 18 , 27 ], six involved only men [ 5 , 13 , 16 , 28 – 30 ], five involved both men and women [ 7 , 9 , 22 , 31 , 32 ] and three studies did not report the sex of the subjects [ 19 , 33 , 34 ]. The age of the subjects ranged from 18 and 49 years; two studies did not report the age of the subjects [ 28 , 33 ]. The samples were from 10 different countries: three studies were led in Poland, two in Spain, two in the United States, two in China, two in Ireland, one in the United Kingdom, one in Australia, one in Belgium, one in Germany and one from Japan. Regarding the characteristics of the sample, eleven studies of the sixteen were conducted on endurance athletes (runners, cyclists, and triathletes). Among these nine studies considering mediumand long-distance runners [ 5 , 13 , 16 , 18 , 22 , 29 , 30 , 32 , 33 ], three studies incorporated physically active subjects [ 27 , 31 , 34 ], one study on triathletes [ 19 ], a study on cyclists [ 7 ], a study included martial arts professionals [ 9 ], and a study on rugby players [ 28 ]. Most of the studies incorporated in this systematic review (81%) used fecal samples to determine changes in the intestinal microbiota. Of these, mainly 75% determined the 16S ribosomal RNA genetic sequence that is commonly used for identification, classification, and quantitation of microbes within complex biological mixtures such as environmental samples and intestinal samples. A minority of four studies (25%) used plasma samples through the protein enzyme-linked immunosorbent assay test to determine markers such as intestinal fatty-acid binding protein (I-FABP) related to mucosal damage, zonulin associated with intestinal permeability, and cortisol, c-reactive protein, and TNFα related to a proinflammatory status. Int. J. Environ. Res. Public Health 2022,19, 9518 5 of 17 Int. J. Environ. Res. Public Health 2022, 19, x 5 of 18 Figure 1. Flowchart of articles through the search process. Table 2 shows the summary of the studies included. This review is focused on data from 513 participants, and the sample size ranged from 4 to 86 subjects. Two of the sixteen studies involved only women [18,27], six involved only men [5,13,16,28–30], five involved both men and women [7,9,22,31,32] and three studies did not report the sex of the subjects [19,33,34]. The age of the subjects ranged from 18 and 49 years; two studies did not report the age of the subjects [28,33]. The samples were from 10 different countries: three studies were led in Poland, two in Spain, two in the United States, two in China, two in Ireland, one in the United Kingdom, one in Australia, one in Belgium, one in Germany and one from Japan. Regarding the characteristics of the sample, eleven studies of the sixteen were conducted on endurance athletes (runners, cyclists, and triathletes). Among these nine studies considering mediumand long-distance runners [5,13,16,18,22,29,30,32,33], three studies incorporated physically active subjects [27,31,34], one study on triathletes [19], a study on cyclists [7], a study included martial arts professionals [9], and a study on rugby players [28]. Most of the studies incorporated in this systematic review (81%) used fecal samples to determine changes in the intestinal microbiota. Of these, mainly 75% determined the 16S ribosomal RNA genetic sequence that is commonly used for identification, classification, and quantitation of microbes within complex biological mixtures such as environmental samples and intestinal samples. A minority of four studies (25%) used plasma samples through the protein enzyme-linked immunosorbent assay test to determine markers such as intestinal fatty-acid binding protein (I-FABP) related to mucosal damage, zonulin associated with intestinal permeability, and cortisol, c-reactive protein, and TNFα related to a proinflammatory status. Figure 1. Flowchart of articles through the search process. The results revealed that nine studies showed negative effects of aerobic exercise on the intestinal microbiota. Among these adverse effects, three studies found an increase in I-FABP [ 5 , 16 , 29 ], one study presented intestinal distress [ 5 ], three studies observed negative changes in the microbiome [ 30 – 32 ], two studies found an increase in Prevotella [ 7 , 33 ], three studies observed an increase in intestinal permeability [ 16 , 19 , 29 ], and two studies reported an increase in zonulin [ 19 , 29 ]. In contrast, seven studies observed positive effects of endurance exercise, including an increase in the level of bacteria such as Roseburia hominis, Bifidobacterium spp., Akkermansia muciniphila,Faecalibacterium prausnitzii [ 18 , 27 ], Coriobacteriaceae [22], increased microbial diversity [9,13,33] and increased intestinal metabolites [28]. Additionally, an analysis has been performed to determine the study qualities included in this review. For this, the Joanna Briggs Institute’s criterium checklist (Table 3) was used. Different criteria were used according to the characteristics of the studies. In this regard, one study fulfilled 100% of the criteria [ 27 ], and seven studies fulfilled ≥ 75% of the quality criteria [ 7 , 9 , 13 , 27 , 28 , 31 , 33 ], classifying themselves as high quality. The rest of the studies [ 5 , 16 , 18 , 19 , 22 , 29 , 30 , 32 , 34 ] were classified as medium quality because they obtained a value between >50% and <75% of the criteria (among 50–69.2%). No studies with low quality were found (<50% of the criteria). Int. J. Environ. Res. Public Health 2022,19, 9518 6 of 17 Table 2. Characteristics as the type of study, aim, sample, design, and mean results of the studies. Author, Year Type of Study AIM Sample Study Design Results Pugh et al. (2017) [5] Quasi-Experimental Characterize the HIIT effects on small intestinal damage markers n= 11 (men runners trained) Aged 33.1 ±10.4; VO2max 60.0 ±3.2 mL/kg/min Acute HIIT episode markers of intestinal permeability and damage were evaluated and compared with resting conditions. Minimum running performance of 10 km (39 min) and a minimum of 5 workout sessions per week, using serum sampling, pre-exercise, after each set of exercises, and 2 h post-baseline HIIT significantly increased the serum lactulose: rhamnose ratio and sucrose concentrations compared with rest. In contrast, urinary lactulose: rhamnose or sucrose concentrations did not vary between study groups. Plasma I-FABP augmented in the recuperation period from HIIT only. After 24 h of HIIT, the researchers found mild symptoms of GI distress Liang et al. (2019) [9] Cross-sectional Whether the intestinal microbiota is distinctive between higher-level and lower-level athletes n= 31 (professional martial arts athletes). 15 women and 16 men; aged 20–24 Martial arts athletes; Wushu routine, vigorous, fast and dynamic sports. The researchers used 16S rRNA gene sequencing to determine the intestinal changes Higher-level athletes have augmented metabolic capacity and diversity in the intestinal microbiota compared with lower-level athletes. Petersen et al. (2017) [7] Cross-sectional Determine the presence of distinctive organisms in professional and amateur level competitive cyclists n = 33 (professional and amateur level competitive cyclists); 11 women and 22 men; aged 19–49 The study used metatranscriptomic (RNA-Seq) sequencing and mWGS The increase in Prevotella was associated with time reported exercising during an average week. Several professional cyclists have augmented levels of Methanobrevibacter smithii transcripts compared with amateur cyclists. Bressa et al. (2017) [27] Cross-sectional Compare intestinal composition among two groups divided by physical exercise levels n= 40 (premenopausal women). 19 active and 21 sedentary Aged 18–40; BMI 20–25 kg/m2 The researchers used 16S rRNA gene sequencing to determine the intestinal changes Performance of physical activity was associated with the presence of health-promoting bacteria (R. hominis,A. muciniphila, Bifidobacterium spp., and F. prausnitzii). Decreased levels of diversity were correlated with sedentary parameters Int. J. Environ. Res. Public Health 2022,19, 9518 7 of 17 Table 2. Cont. Author, Year Type of Study AIM Sample Study Design Results Karhu et al. (2017) [16] Quasi-experimental Evaluate the effect of running on GI function markers n= 17 (active runners); 8 women and 9 men; aged 18–45 The researchers measured secondary variables, such as zonulin, levels of serum intestinal I-FABP, and bacterial LPS, among others Both, serum I-FABP and intestinal permeability increased after running, without differences amongst groups. No changes were observed in the bacterial LPS in serum Keohane et al. (2019) [13]Long-term Analyze the changes in the intestinal microbiota of four well-trained male athletes to prolonged, high-intensity trans-oceanic rowing n= 4 (men athletes transatlantic rowing). Aged 25–27; BMI 23–25 kg/m2; VO2Max 46–50 mL/kg/min Metagenomic whole-genome shotgun sequencing was used Intense exercise clearly impacts the diversity of the intestinal microbiota, with changes in specific bacteria related to metabolic pathways Bycura et al. (2021) [31] Quasi-experimental Impact of CRE or RTE on intestinal microbiota n= 56 n= 28 CRE group (21 women; Aged 20.7; BMI 24.5 kg/m2and 7 men; aged 20; BMI 24.0 kg/m2.n= 28 RTE group (17 women; aged 20.4; BMI 23.2 kg/m2and 11 men; aged 22.6; BMI 24.59 kg/m2 Intestinal microbiota was measured using 16S rRNA gene sequencing The observed changes were associated only with the CRE group, resulting in disturbance of the intestinal microbiota Morishima et al. (2020) [18]Cross-sectional Effects of highly intensive endurance exercise on the intestinal microbiota and its relationship with the onset of the exercise-induced GI disorders n= 29 (15 women Japanese endurance runners and 14 nonathletic but healthy women). Aged 20–21; BMI 20.7–21.9 kg/m2 Fecal microbiota was tested using 16S rRNA metagenomics, and other variables such as moisture content, organic acids, and putrefactive metabolites concentrations were examined Female elite endurance runners have more abundance of Faecalibacterium, and these changes could be associated with the succinate concentration in this group Tota et al. (2019) [19] Long-term Evaluate intestinal and muscle damage in triathletes n= 15 (triathletes). Aged 6–14; VO2max 58.8 ±4.5 mL/kg/min Variables used for the analysis were: cortisol, c-reactive protein, zonulin, and TNF-α Zonulin and variables of permeability were augmented after the race Int. J. Environ. Res. Public Health 2022,19, 9518 8 of 17 Table 2. Cont. Author, Year Type of Study AIM Sample Study Design Results Zhao et al. (2018) [22] Quasi-experimental The gut microbiota immediately responds to the enteric changes in amateur half-marathon runners n= 20 (4 women and 16 men amateur half-marathon runners). Aged 31.3; BMI 22.6 kg/m2 Fecal samples were analyzed before and after the marathon using 16 rDNA sequencing analyses Coriobacteriaceae changes were related to the exercise role in avoiding disease and refining health outcomes. Moitinho-Silva et al. (2021) [34] Randomized controlled trial Analyze the changes in the intestinal microbiota on previously physically inactive, healthy adults in comparison to controls that did not perform regular exercise n= 36 (11 controls; 13 endurance group; 12 strength group). Aged 22–41.3; BMI 19.7–32.5 kg/m 2 Fecal microbiota was tested using 16S rRNA metagenomics Mucosal damage and inflammation were found after short-term resistance training. No changes were observed in intestinal microbiota Sadowska-Krepa et al. (2021) [29]Quasi-experimental Evaluate intestinal damage in middle-aged male subjects n= 10 (amateur long-distance runners). Aged 21–35 Variables used for the analysis were: TAS, TOS/TOC, hs-CRP, I-FABP, and zonulin After the exercise, the levels of intestinal permeability biomarkers as, hs-CRP, I-FABP, zonulin, and inflammation were augmented Kulecka et al. (2020) [33]Quasi-experimental Evaluate differences in intestinal microbiota amongst healthy controls and endurance athletes n= 71 n= 14 marathon runners; n= 11 cross-country skiers; n= 46 healthy control individuals Fecal microbiota was tested using 16S rRNA metagenomics Excessive training is associated with changes in Bacteroides and Prevotella and bacterial diversity Tabone et al. (2021) [30] Quasi-experimental Determine whether the changes are driven by exercise on the gut microbiota (with 16S rRNA gene) and the serum and fecal metabolome n= 40 (men endurance cross-country runners). Aged 35.8 ±8.0; BMI 22.8 ±2.1 kg/m2; VO2max 58.8 ±3.24 mL/kg/min Fecal microbiota was tested using 16S rRNA metagenomics The changes in gut microbiota could be related to physiological changes in ammonia, uric acid, and lactate Int. J. Environ. Res. Public Health 2022,19, 9518 9 of 17 Table 2. Cont. Author, Year Type of Study AIM Sample Study Design Results Barton et al. (2017) [28] Cross-Sectional Evaluate differences in intestinal microbiota amongst exercise and a more sedentary state n= 86 (40 men professional international rugby union players and 46 men controls) Fecal microbiota was tested using 16S rRNA metagenomics Professional international rugby union players had more favorable effects in metabolic pathways than the control group Craven et al. (2021) [32] Quasi-experimental Evaluate differences in intestinal microbiota according to training volume n= 14 (highly trained middle-distance runners). n= 6 women; aged 22.0 ± 3.4; VO2max 59.0 ±3.2 mL/kg/min n= 8 men; aged 20.7 ±3.2; VO2max 70.1 ±4.3 mL/kg/min Fecal microbiota was tested using 16S rRNA metagenomics No changes were observed in intestinal microbiota according to training volume in upper taxons. Changes in family, genus, and species were observed, these changes did not return to pre-levels Abbreviations: AFT, after fecal; BEF, before fecal; BMI, body mass index; CRE, cardiorespiratory exercise; DS, standard deviation; FCCS, female cross-country skiers; FDR, false discovery rate; FMR, female marathon runners; GI, gastrointestinal; HIIT, high-intensity interval training; hs-CRP, High-sensitivity C-reactive protein; HvolTr, high-volume training; I-FABP, intestinal fatty acid-binding protein; kg/m 2 , kilogram per square meter; LPS, lipopolysaccharide; MCCS, male cross-country skiers; MCHC, mean corpuscular hemoglobin concentration; mL/kg/min, milliliters per minute per kilogram; MMR, male marathon runners; mWGS, metagenomic whole genome shotgun; NormTr normal training; PGM, personal genome machine; PWC, physical working capacity; rRNA, ribosomal ribonucleic acid; RTE, resistance training exercise; TaperTr, exponential reduction in training; TAS, total antioxidant status; TOC, total oxidant capacity; TOS, total oxidant status; VO2max, the maximum amount of oxygen; WHO, World Health Organization; WSER, Western States Endurance Run. 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