Effects of Probiotic Supplementation on Sports Performance and Performance-Related Features in Athletes: A Systematic Review

ew aims to evaluate the effects of probiotic supplementation on performance and performance-related conditions in athletes by evaluating randomized controlled studies from the MEDLINE (Pubmed), Web of Science, Scopus, and SPORTDiscus (EBSCO) databases. From a total of 2304 relevant articles, 13 studies ful lled the inclusion criteria. Seven studies concern endurance athletes, one to rugby players, three refer to non-speci ed athletes, one to badminton players, and one involves baseball players. The evidence suggests that the integration of athletes' diets with some bacterial strains and also the consumption of multi-strain compounds may lead to an improvement in performance and can positively affect performance-related aspects such as fatigue, muscle pain, body composition, and cardiorespiratory tness. However, the type of supplementation and sport is very variable among the studies examined. Therefore, to obtain more solid evidence, further controlled and comparable studies are needed to expand the research regarding the possible repercussions of probiotics use on athletes' performance.

improvement in performance and can positively affect performance-related aspects such as fatigue, muscle pain, body composition, and cardiorespiratory tness. However, the type of supplementation and sport is very variable among the studies examined. Therefore, to obtain more solid evidence, further controlled and comparable studies are needed to expand the research regarding the possible repercussions of probiotics use on athletes' performance. Keywords:athletes; sport; performance; probiotic; diet supplementation 1. Introduction It is known that physical activity (PA), which includes any form of movement in which the contraction of skeletal muscles results in an increase in energy consumption, has numerous bene cial effects on human health [1]. Regularly performing endurance and muscle-strengthening PA can improve physiological parameters which in turn lead to an enhancement in health-related physical tness, a physiologic state of well-being related to health status including cardiovascular tness, musculoskeletal tness, body composition, and metabolism [1,2]. PA is also able to maintain or improve human neurocognitive conditions, and strengthen immune defences [2]. Scienti c evidence has underlined the preventive role of regular physical activity towards chronic diseases such as diabetes, cancer, and cardiovascular diseases and related premature death [1,2]. Physical exercise is aimed to improve body functionality through adaptation. Current evidence suggests that physical exercise can affect, in quantitative and qualitative terms, the intestinal microbiota composition. It seems, in fact, that physical exercise generates greater microbial diversity in the gut, increases the Bacteroidetes-Firmicutes ratio, stimulates the proliferation of bacteria that can modulate mucosal immunity, and improves the intestinal barrier functions, with bene cial effects on the health of the host [3–12]. However, it has been observed that high-intensity exercise, which is common in individuals practising sports, can have detrimental effects on health. It can cause an increase in intestinal permeability and a decrease in the thickness of the intestinal mucus, potentially allowing pathogens/toxins to enter the bloodstream; moreover, it has been associated with immunosuppression by decreasing the function of immune cells, which Int. J. Environ. Res. Public Health2023,20, 2226.

decrease in the thickness of the intestinal mucus, potentially allowing pathogens/toxins to enter the bloodstream; moreover, it has been associated with immunosuppression by decreasing the function of immune cells, which Int. J. Environ. Res. Public Health2023,20, 2226.

Int. J. Environ. Res. Public Health2023,20, 2226 2 of 14 improves susceptibility to infections [13–19], such as upper respiratory tract infection (URTI) [19–21]. This can be related to acute immune failure and chronic suppression of immune factors which follow frequent and strenuous exercise [22,23]. In addition, during intense training and competitions, gastrointestinal (GI) disorders, such as diarrhoea and heartburn [17,18], can occur. Causing interruptions in training or competitions, the aforementioned pathologies can have a negative indirect impact also on athletic performance [24,25]. Therefore, the reduction of these effects on athletes becomes a top priority. In recent years, there are growing studies supporting the ef cacy of taking probiotics, microorganisms which can have bene cial effects on the organism, in reducing the inci- dence and severity of acute infectious diarrhoea and Upper Respiratory Tract Infections (URTIs) in the general population [26–28]. These effects derive from the modulation of the immune system operated by probiotics. Furthermore, probiotics are also modulators of the intestinal microbiota, which can also have an indirect in uence on various indices of physi- cal performance and subsequent recovery. A recent systematic review has demonstrated the bene cial effects of probiotics in reducing the risk of developing GI and respiratory infectious diseases or the severity of symptoms associated with these disorders also in athletes [29]. Therefore, supplementation with probiotics could have positive effects on athletes' health [25,30]. Besides the evidence regarding the effects on the athletes' immune defences, it is not clear, however, if and which type of probiotic supplementation may lead to bene cial effects on sports performance in athletes. In particular, its possible effects on all the major dimensions of sports performance (skill, strength, endurance, and recovery) should be investigated from this point of view. Furthermore, since physical tness provides the basis for sports performance [1], it is interesting to assess which performance-related physiological parameters can be affected by probiotic supplementation in athletes. To address these questions, this review aimed to critically analyze the literature regarding the effects of probiotics supplementation on performance and performance- related conditions and characteristics in athletes. 2. Materials and Methods The systematic

physical tness provides the basis for sports performance [1], it is interesting to assess which performance-related physiological parameters can be affected by probiotic supplementation in athletes. To address these questions, this review aimed to critically analyze the literature regarding the effects of probiotics supplementation on performance and performance- related conditions and characteristics in athletes. 2. Materials and Methods The systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [31]. The Review protocol was registered on PROSPERO (CRD42021268105). 2.1. Eligibility Criteria The selection of the studies was performed through the PICO model, by consider- ing the following parameters: P (patients) healthy adult athletes; I (intervention) diet supplementation with probiotics; C (comparison) comparison with a control group; O (out- comes) potential effects of probiotic supplementation on performance. Only randomized controlled studies performed on healthy adult athletes with a description of probiotics supplementation were included. The inclusion and exclusion criteria are shown in Table. Table 1.Inclusion and exclusion criteria used in the article selection. Inclusion Criteria Exclusion Criteria Randomized controlled studies Studies on healthy adult athletes Studies that evaluated probiotics' effects on performance and/or on performance-related physiological parameters Studies based on probiotic supplementation as an intervention Studies reporting the type and dose of probiotic supplementation Studies on animals Studies on non-healthy individuals Studies on non-physically active individuals Studies performed on children Studies not reporting supplementation details

Int. J. Environ. Res. Public Health2023,20, 2226 3 of 14 2.2. Literature Search and Selection of Studies The article search was carried out by keywords (probiotic AND sport OR exercise OR athletes OR physical activity). Further articles were also searched in the reference lists of available reviews. Articles in English, Spanish, Italian and French languages were considered. The search was completed at the end of July 2022. 2.3. Data Collection The articles found by database search were independently assessed by two reviewers (M.D.D and P.C.). Duplicate articles across the different databases were excluded, and then potentially eligible studies were identi ed by title and abstract screening. The same reviewers analyzed independently the full texts of these articles and selected those studies that met the selection criteria. Another reviewer (V.D.O.) resolved the disagreements. Two other reviewers (G.C. and C.P.P.) extracted the following information from each selected study: authors and year of publication, study design, sample size and demographic characteristics of participants, characteristics of probiotic supplementation, outcomes and main results concerning sports performance and physical conditions or characteristics related to performance. 2.4. Risk of Bias The revised Cochrane Risk-of-Bias tool for randomized trials (RoB2) was used to assess the risk of bias in the selected studies [32]. The evaluation was performed by two reviewers (M.D.D. and P.C.). Con icts were resolved by a third researcher (F.G.). The risk of bias for each study was de ned as low, moderate, or high. 3. Results 3.1. Article Selection and Characteristics A total of 2304 relevant articles were initially found (Figure). Of these, 403 were considered eligible. After eliminating duplicates from the different databases and non- randomized studies, 13 articles [33–45] were selected considering the inclusion and exclu- sion criteria (Table). The selected studies were performed between 2014 and 2021 in different geographic areas: Europe (Austria, UK, Spain, Poland), West Asia (Israel), East Asia (Malaysia, Japan, Taiwan), North America (USA, Tennessee) and Oceania (Australia). The majority of them had a double-blind, placebo-controlled design. Table participants' gender [34,38,43], eight studies involved only males [33,35–37,39–41,45], one study involved only females [44] and one study included both genders

performed between 2014 and 2021 in different geographic areas: Europe (Austria, UK, Spain, Poland), West Asia (Israel), East Asia (Malaysia, Japan, Taiwan), North America (USA, Tennessee) and Oceania (Australia). The majority of them had a double-blind, placebo-controlled design. Table participants' gender [34,38,43], eight studies involved only males [33,35–37,39–41,45], one study involved only females [44] and one study included both genders [42]. The mean age was included between 19.5 1.0 and 37.21 8.9 years. Seven studies concern endurance athletes [34,35,37,39–42], one study concerns rugby players [33], three studies refer to non-speci ed categories of athletes [36,43,44], one study concerns badminton players [38] and one study involved subjects who played baseball [45]. In seven studies [34–36,38,39,44,45] the probiotic supplementation was represented by a single bacterial strain, while in the remaining studies, it included the consumption of a multi-strain compound. Regarding the risk of bias assessment, eight out of thirteen studies showed a low risk of bias (Table).

Int. J. Environ. Res. Public Health2023,20, 2226 4 of 14Int. J. Environ. Res. Public Health 2023, 20, x FOR PEER REVIEW 4 of 16 Figure 1. Prisma flow diagram of the article selection. The selected studies were performed between 2014 and 2021 in different geographic areas: Europe (Austria, UK, Spain, Poland), West Asia (Israel), East Asia (Malaysia, Ja- pan, Taiwan), North America (USA, Tennessee) and Oceania (Australia). The majority of them had a double-blind, placebo-controlled design. Table 2 shows the characteristics of each study. Three studies did not report the participants’ gender [34,38,43], eight studies involved only males [33,35–37,39–41,45], one study involved only females [44] and one study included both genders [42]. The mean age was included between 19.5 ± 1.0 and 37.21 ± 8.9 years. Figure 1.Prisma ow diagram of the article selection.

Int. J. Environ. Res. Public Health2023,20, 2226 5 of 14 Table 2.Characteristics of the included studies. Author, Year, Country, Study Design Sample Characteristics N of Subjects_ M/F_ Mean Age Probiotics Daily Intake Intervention Length Type of Performance/Physical Condition Variables Studied Main Results Harnett, 2020 Australia -double-blind randomised controlled trial Elite male rugby union players Probiotic group (n= 9) 27.0 3.2 years Placebo group (n= 10) 26.6 2.9 years generaLactobacillus, Bi odbacteriumand Streptococcus(during international competition) Dose: 60 bilion SBFloractivTM (Bioceuticals, Australia AustL# 285024) containing 250 mg of the yeastSaccharomyces boulardiiwas added to the probiotic regime during the international travel 17 weeks Muscle soreness rated on a 1–5 scale: 5 = non-existent, 4 = ne, no dramas let's get on with it, 3 = there's some, but after normal warm-up, I'll be ne, 2 = pretty ordinary, my movements are stiff and sore, and1 = severe, I need to alert the physio or doctor. Leg heaviness on a 1–10 Likert scale Muscle soreness was 0.5 units lower (F(1, 343) = 42.646,p< 0.0001) and leg heaviness scores 0.7 units lower (F(1, 334) = 28.990,p< 0.0001) in the probiotic group compared to the placebo group. Across both groups, as self-reported muscle soreness scores and salivary CRP (C-reactive protein) concentrations increased, sleep quantity, quality and motivation scores decreased. Conversely, as muscle soreness scores and CRP decreased, sleep quantity and quality, and motivation scores improved. Ching Huang, 2019 Taiwan -double-blind experimental design Triathletes 18 subjects for Study I 16 subjects for study II Probiotic group (n= 9) STUDY I 20.2 0.7 years STUDY II 22.3 1.2 years Placebo group (n= 9) STUDY I 21.1 1.5 years STUDY II 20.1 0.3 years LyophilizedL. plantarumPS128 Dose: twice capsules per day (3 10 10 CFU/day) 4 weeks (STUDY I) 3 weeks (STUDY II) Body composition evaluated using DEXA (dual-energy X-ray absorptiometer). Anaerobic and aerobic capacities evaluated using a 30-s Wingate anaerobic kinetic Test and VO2 max endurance test. Muscle damage evaluated using Biochemical Indices, such as CK, LDH, protein carbonyl, myioglobin. Muscle fatigue evaluated using Biochemical Indices, such as Ammonia, Lactate and FFA. L. plantarumPS128 supplementation, combined with training,

3 weeks (STUDY II) Body composition evaluated using DEXA (dual-energy X-ray absorptiometer). Anaerobic and aerobic capacities evaluated using a 30-s Wingate anaerobic kinetic Test and VO2 max endurance test. Muscle damage evaluated using Biochemical Indices, such as CK, LDH, protein carbonyl, myioglobin. Muscle fatigue evaluated using Biochemical Indices, such as Ammonia, Lactate and FFA. L. plantarumPS128 supplementation, combined with training, can signi cantly alleviate oxidative stress (such as creatine kinase, Thioredoxin, and Myeloperoxidase indices) after a triathlon (p< 0.05). This effect is possibly regulated by a 6–13% decrease of indicated pro-in ammation (TNF- , interleukin-6, and interleukin-8) cytokines (p< 0.05) and 55% increase in anti-in ammation (interleukin-10) cytokines (p< 0.05) after intensive exercise stimulation. In addition,L. plantarumPS128 can also substantially increase 24–69% of plasma-branched amino acids (p< 0.05) and elevate exercise performance, as compared to the placebo group (p< 0.05). There was no signi cant difference in body composition between the probiotic group and placebo group pre- and post-supplementation (p 0.05). Ching Huang, 2020 Taiwan -double-blind experimental design Male Triathletes Probiotic group (n= 10) 21.9 1.4 years Placebo group (n= 10) 21.6 1.3 years L. plantarumPS128 Dose: a single capsule twice per day, equivalent to 3 10 10 CFU/day 4 weeks Maximal oxygen consumption and exercise performance evaluated using a treadmill (Pulsar, h/p/cosmos, Germany) and an auto respiratory analyzer K4b2 (Cosmed, Concord, CA, USA). Body composition evaluated using DEXA (dual-energy X-ray absorptiometer). L. plantarumPS128 supplementation was associated with an improvement in endurance running performance through microbiota modulation and related metabolites, but not in maximal oxygen uptake. The probiotic group could signi cantly elevate endurance performance by the treadmill exercise protocol; the performance could increase by about 130% as compared to the placebo group (p= 0.0035). However, at the end of the study, the VO2 max and body composition (bone, fat, and lean percentage) demonstrated no signi cant difference between groups in the gas and DEXA analysis.

Int. J. Environ. Res. Public Health2023,20, 2226 6 of 14 Table 2.Cont. Author, Year, Country, Study Design Sample Characteristics N of Subjects_ M/F_ Mean Age Probiotics Daily Intake Intervention Length Type of Performance/Physical Condition Variables Studied Main Results Komano, 2018 Japan -randomized, placebo-controlled, double-blinded trial Healthy male athletes Probiotic group (n= 26) 20.8 0.8 years Placebo group (n= 24) 20.5 0.8 years cells of heat-killedLactococcus lactis strain plasma Dose: a capsule containing 100 billion cells 13 days Physical condition, fatigue, articular pain, lassitude, and muscle pain evaluated using a daily questionnaire. CD86 (Cluster of Differentiation 86) as a maturation marker on pDC (plasmacytoid dendritic cells) was signi cantly increased in the probiotic group. Moreover, the cumulative days of fatigue were signi cantly fewer in the probiotic group. Pugh, 2020 UK -randomized, double-blind, placebo-controlled crossover trial Trained male cyclists (n= 7) 23 4 years Active strainsLactobacillus acidophilus (CUL60),Lactobacillus acidophilus (CUL21),Bi dobacterium bi dum(CUL20), andBi dobacterium animalissubsp.lactis (CUL34; Proven Probiotics, Port Talbot, UK) During exercise, subjects consumed a 10% CHO drink enriched with the stable isotope [U-13C] glucose (CK Isotopes, Ibstock, UK). Maltodextrin (176.4 g; Myprotein Inc., Northwich, UK) and 3.6 g [U-13C] glucose Dose: a capsule containing 25 billion CFU-two, 28-day, periods of supplementation, separated by a 14-day washout period. Exercise trials made using time trial Probiotics led to minimal increases in absorption and oxidation of the ingested maltodextrin and small reductions in fat oxidation, whereas having no effect on subsequent time-trial performance. During the 100-kJ time trial, there was no signi cant difference in the time to complete between placebo group (308 69 s) and probiotic group (301 74 s;p= 0.714). Salleh, 2021 Malaysia -randomized, placebo-controlled study Badminton Players Probiotic group (n= 15) 19.5 1.0 years Placebo group (n= 15) 19.9 1.3 years Lactobacillus casei Shirota Dose: drink containing 3 10 10 CFU 6 weeks Body composition evaluated using the InBody 500 bioelectrical impedance analyser. Aerobic Capacity evaluated using a 20-m multi-stage shuttle run test. Hand strength evaluated using the handgrip test. Leg power assessment measuring the distance of the most extreme point the subject could reach with their arm

19.9 1.3 years Lactobacillus casei Shirota Dose: drink containing 3 10 10 CFU 6 weeks Body composition evaluated using the InBody 500 bioelectrical impedance analyser. Aerobic Capacity evaluated using a 20-m multi-stage shuttle run test. Hand strength evaluated using the handgrip test. Leg power assessment measuring the distance of the most extreme point the subject could reach with their arm by jumping. Speed evaluated using a 40-m dash. Agility evaluated using a t-test. Supplementation of probiotics improved aerobic capacity in probiotic group by 5.9% (p< 0.001) but did not in uence the speed, strength, leg power and agility.

A systematic review evaluating the impact of probiotics on athletic performance.