The Effects of Probiotic Supplementation on Performance, Body composition, Gastro- intestinal Symptoms and Mood Status in Unprofessional Athletes: Study Protocol for a Randomized Double-blind Clinical Trial

The Effects of Probiotic Supplementation on Performance, Body composition, Gastro- intestinal Symptoms and Mood Status in Unprofessional Athletes: Study Protocol for a Randomized Double-blind Clinical Trial | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Effects of Probiotic Supplementation on Performance, Body composition, Gastro- intestinal Symptoms and Mood Status in Unprofessional Athletes: Study Protocol for a Randomized Double-blind Clinical Trial Nastaran Mahmoudi Shirkoohi, Alireza Chegol, Hamed Mohammadi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6712303/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Probiotic supplementation has emerged as a promising strategy in sport nutrition, with potential benefits for various aspects of athletes' health and performance. The study aims to explore the impact of probiotic supplementation on performance, body composition, gastrointestinal symptoms, and mood status in non-elite athletes. Methods/Design: In this double-blind, randomized controlled clinical trial, 36 non-elite athletes were randomly assigned to receive a probiotic supplement (containing 2×10 9 colony-forming units Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei) or identical placebo once a day for 12 weeks. The body composition, muscle strength, and performance will be measured at baseline and following supplementation. The sociodemographic, physical activity and dietary intake questionnaires will be collected for each subject. Additional questionnaires will inquire about GI symptoms, and mood status at baseline and following treatment. The evaluation of primary and secondary outcomes will involve comparing results among intervention groups, considering baseline values. Discussion: By promoting a healthy balance of gut microbiota, probiotics may offer a natural and effective way to improve overall well-being and enhance various aspects of performance. This trial’s findings may be incorporated in to evidence-based guidelines that address the alternation of gut microbiota to enhance athletic performance. Ethics and dissemination: There are so far no reported adverse effects associated with the use of probiotic. The research has obtained approval from the Medical Ethics Committee at Tehran University of Medical Sciences in Iran ethical code: IR.TUMS.MEDICINE.REC.1403.157 Trial registration: IRCT20240424061562N1, registered on 30/7/2024. https://www.irct.ir/trial/77722. Health sciences/Health care/Nutrition Biological sciences/Psychology Probiotic supplementation Gastrointestinal symptoms Mood states Exercise performance Unprofessional Athlete Figures Figure 1 Figure 2 What is already known about this topic? Probiotic supplementation is known to improve gut health by enhancing microbiota diversity, which can alleviate gastrointestinal symptoms common in athletes. Additionally, there is evidence suggesting that probiotics may enhance exercise performance through better nutrient absorption and digestion, support immune function to reduce illness risk, and positively impact mood and mental well-being, potentially alleviating anxiety and depression. Some strains may also assist in body composition management, yet research findings vary due to factors like strain specificity and individual differences. Most existing studies focus on professional athletes, highlighting a gap in understanding the effects of probiotics on unprofessional athletes, which the current study aims to address. What does this article add? This article adds significant value by focusing on unprofessional athletes, a demographic often neglected in probiotic research. It employs a rigorous randomized double-blind clinical trial design to provide a comprehensive evaluation of probiotic supplementation, examining its effects on performance, body composition, gastrointestinal symptoms, and mood status. By specifically investigating three commonly used probiotic strains among athletes, the study aims to identify which formulations may be most beneficial. The findings could lead to practical dietary recommendations, helping unprofessional athletes optimize their health and performance. Overall, this research enhances the understanding of probiotics in athletic contexts. Introduction Background and rationale Athletes are always looking for ways to improve their performance. The success of an athlete is dependent on characteristics such as genetic, physiological, and psychological characteristics, and on the other hand, it is dependent on targeted and scientific planning in the framework of regular and organized training and nutrition (1). Fatigue, mood disturbances, and gastrointestinal distress are the most important obstacles for athletes to achieve better performance (2). Therefore, due to the desire of athletes to achieve their athletic goals quickly, they often resort to supplements, energy boosters, and occasionally even misuse medications (3). The consumption of various supplements is observed not only among professional athletes, but also among beginner athletes and non-athletes (4). Additionally, research indicates a high prevalence of supplement usage among Iranian athletes, standing at 64.8% (5). But one of the supplements that is less commonly used by athletes is probiotics. Probiotics are often overlooked supplements that are crucial for both health and enhancing athletes’ performance (6). According to the classification of sports supplements by the sports nutrition department of the Australian Institute of sports (AIS), probiotics are class A (first) supplements, so it seems that this supplement can have an increasing effect on improving sports performance (7). The International Olympic Committee (IOC) introduces probiotics as useful living microorganisms for gut health and immune system modulation (8). Studies have highlighted the beneficial impact of probiotics on enhancing the immune system post intense exercise, alleviating digestive issues through microbiota modulation and SCFA production, mitigating respiratory symptoms, and lowering the susceptibility to respiratory infections (9, 10). In addition, intestinal dysbiosis activates the hypothalamic-pituitary-adrenal axis (HPA), leading to depression and anxiety. Probiotics can improve gut dysbiosis by suppressing this overactivated axis, which decreases serotonin and increases inflammatory cytokines (11-13). Numerous researchers have highlighted the advantages of probiotic use in enhancing athletic performance (14). Mazur-Kurach et al. in 2022 (15) investigated the role of probiotics in the sports performance of road cyclists. The findings showed that probiotics increased maximal oxygen consumption (VO 2max ), training time to exhaustion, load on the ergometer, and the degree of heart rate reduction. Despite its impact on athletes’ aerobic capacity, probiotics did not show any noticeable effects on anaerobic capacity or body composition. In an investigation conducted by Salleh et al (16) among 30 badminton players, the results showed 6 weeks of supplementation with probiotics significantly increased the aerobic capacity of athletes. Also, the consumption of this supplement led to a reduction in the level of stress and anxiety and improved mood in athletes. In a study by Schreiber et al. (17) on elite cyclists over a 90-day period, probiotics were examined for their impact on sports performance, inflammatory markers, and gastrointestinal symptoms. The study results revealed no significant disparities in VO 2max , maximum heart rate, or time to fatigue between the intervention and placebo groups. However, the findings did demonstrate that probiotic supplementation led to improvements in digestive symptoms among the participants. Probiotic treatment might affect physiological responses to exercise greater among unprofessional athlete, as well as immunomodulatory and gastrointestinal benefits that could improve performance and benefit athletes (18). Thus, the goals and hypotheses of this double-blind, randomized placebo-controlled clinical trial will be as follows. Objectives This study’s main goal is to evaluate the ergogenic effects of probiotic supplementation on anaerobic performance in unprofessional athletes. Secondary objectives include assessment of body composition, gastro-intestinal symptoms, mood status, and muscle strength. Trial design A randomized, parallel, double-blind and placebo-controlled clinical trial will be implemented. Methods: participants, interventions, and outcomes Study setting The current study is a placebo-controlled, randomized, double-blind clinical trial that explore the impacts of probiotic use in non-professional athletes. This study was conducted according to the Helsinki Declaration and written informed consent was obtained from all participants. The research has obtained approval from the Medical Ethics Committee at Tehran University of Medical Sciences in Iran ethical code: IR.TUMS.MEDICINE.REC.1403.157 and is already registered in the Iranian Registry of Clinical Trials under the ID IRCT20240424061562N1. The study will be presented following the CONSORT 2010 guidelines. Eligibility criteria All participants were included in the current trial if met our inclusion criteria: 1) healthy adults aged 20–40 years old who have an average of 6–10 hours of training per week; 2) a history of at least 3 months of bodybuilding training; 3) athletes who consent to participate in this study by signing a formal informed consent form. Non-inclusion criteria were as follows: 1) professional athletes who practice for more than 6 hours a week; 2) pregnancy and lactating; 3) premature menopause; 4) Various medical conditions can impact individuals’ capacity to participate in or partake in physical activity; 5) frequent intake of probiotic products, for example dietary fibers such as inulin and galacto-oligosaccharides, regularly 1 month before and during the study; 6) smokers who are currently in the habit, suspected or confirmed history of alcohol or drug misuse; 7) consumption of food and sports supplements at least four to six weeks prior to and during the intervention; 8) adhering special diets; 9) taken any antibiotics during the course of the treatment or in the past four weeks; 10) having gastrointestinal diseases, i.e., food allergies, celiac, irritable bowel disease, Whitin the last two months; 11) engaging in sports other than bodybuilding; 12) having acute gastrointestinal symptoms up to two months before the study. Exclusion criteria: 1) Allergy to studied agents 2) Noncompliance in consumption of the supplements (<90%) 3) taking antibiotics during the study 4) pregnancy during study. Informed consent At baseline, the trial’s goal will be explained to the legal guardian by the principal investigator (N.M), who will obtain their written informed consent upon enrollment. Also, they will be assured that their information will remain confidential ( Appendix1 ). Interventions Thirty-six eligible participants will be randomly assigned to either the placebo group (n = 18) or the intervention group (n = 18). Participants in both groups will be administered probiotic capsules containing Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei (2 billion colony-forming units), along with identical placebo capsules containing 100 mg of maltodextrin daily with a meal for a duration of 12 weeks. The Tak Gen Zist Pharmaceutical Company, Tehran, Iran, will manufacture the intervention and control boxes. They will be equivalent in size, taste, smell, and package (tagged 1 and 2), allowing for allocation concealment. Two separate microbiologists verified microbiological and purity tests. Any clinical adverse event (AE) linked to the intervention, laboratory abnormalities, or other medical conditions (such as pregnancy) that would not benefit the participant’s well-being will be grounds for discontinuing the intervention in the study. All supplements and tests in this program will be provided to athlete free of charge, and during the three months they patriciate in this study, they will receive free diet by nutritionist. Outcomes: The primary outcome assesses alterations in RAST (Running-based anaerobic sprint test) performance metrics, such as minimum, average, peak power, and fatigue index, in the probiotic and placebo groups after a 12-week intervention compared to baseline. Secondary outcomes, involving a comparison of the probiotic and placebo groups while accounting for their respective baseline values, include the following: 1) changes in body composition and anthropometric indices (weight, body mass index (BMI), waist circumference (WC), fat-free mass (FFM), fat mass (FM), lean body mass (LBM), a body shape index (ABSI), body roundness index (BRI), waist-to-hip ratio (WHR), body adiposity index (BAI), abdominal volume index (AVI), conicity Index (ConI), and waist-to-height ratio (WHtR)) from baseline; 2) gastrointestinal symptoms that will be evaluated through the Gastrointestinal Symptoms Rating Scale (GSRS) questionnaire at the beginning and end of the study; 3) mood status that will be evaluated through Brunel Mood Scale (BRUMS) questionnaire at the beginning and end of the study; 4) changes in muscle strength Participant timeline The timeline of the trial and study flow chart of enrollment, allocation, intervention, and assessment are presented in Table 1 and Fig. 1 , respectively. Initially, all participants who consent will undergo a 2-week run-in phase, during which three 24-hour food recalls (two on working days and one on a weekend) will be conducted to collect dietary information. Demographic details, physical activity levels, medical history, medication usage, and socioeconomic status data will be collected through a questionnaire and face-to-face interviews. Following the run-in phase, assessments will be made on body composition, GI symptoms, mood status, and performance. Subsequently, participants will be categorized by gender (male/female) into blocks and then randomly assigned to either the intervention or control groups. After 6 weeks, participants will undergo another round of 24-hour food recalls. Finally, following the conclusion of the study, all outcomes will be reassessed. Sample size The sample size was determined to be 16 for each group using a two-sided t-test, considering a type I error of 5% (α = 0.05), an 80% power, and sport performance as the primary outcome (19). To accommodate an anticipated 10% dropout rate during the study, the final sample size was increased to 18 participants per group. Recruitment The recruitment for this study is set to commence in August 2024 and is anticipated to conclude in February 2024. Participants are invited to participate in this project based on the advertisement in social networks, sports clubs and sports medicine clinics. If they wish to cooperate, they will be informed about the implementation of the project in general, then we will provide the necessary explanations about the probiotic supplement and the purpose of the study to the people, then informed written consent will be obtained from the participants. Randomization, sequence generation, concealment, and blinding: Participant randomization to the intervention/placebo sequence will be carried out by a local pharmacist utilizing permuted-block randomization stratified by gender (block sizes 4, 6, 6) through the designated website: https://www.sealedenvelope.com/simple-randomiser/v1/lists [Accessed 2 Aug 2024]. The experimental and placebo nutritional supplements will be dispensed to participants by the pharmacist in the specified sequence. The pharmacist’s role will solely involve providing the supplements, without any direct interaction with the study participants or involvement in data collection or processing. Opaque and sealed containers containing dietary supplements will be distributed monthly and monitored for compliance by weighing. External pharmaceutical packaging services will handle the off-site packaging of supplements. Each package will bear a participant’s study identification number and be labeled as Supplement 1 or Supplement 2 based on the intervention phase to ensure treatment concealment from volunteers and investigators. Data collectors and data administrators will be blinded to the group assignment. Unblinding will only occur after the completion of statistical analysis of the data or if serious adverse events occur, suspicious unintended serious adverse reactions, it can be uncovered after asking the leading researcher. Furthermore, supplement delivery to participants will occur on the 1st, 30th, and 60th days according to the pre-determined allocation. Data collection and management Assessment of physical activity The study will involve assessing physical activity levels using the abbreviated version of the International Physical Activity Questionnaire (IPAQ)(20). Measurements will be reported in MET-min per week on the IPAQ form. A scoring method based on IPAQ will classify participants into low, moderate, or high activity level groups (21). Throughout the study, adjustments in physical activity levels will be considered during statistical analysis to address potential confounding factors. Anthropometric and body composition assessments During the trial, height and body weight will be measured using the Seca scale and stadiometer with precision to the nearest 0.1 cm and 0.1 kg, respectively. Participants will be required to be barefoot and dressed comfortably for these measurements. Body Mass Index (BMI) will be calculated by dividing weight in kilograms by the square of height in meters. Waist circumference (WC) will be determined by measuring the greatest gluteal muscle, the middle of the lowest rib, and the highest point of the iliac crest. Hip circumference will be measured at the widest point over the buttocks with a measuring tape accurate to 0.1 cm. The Waist-to-Hip Ratio (WHR) can be calculated by dividing the waist circumference by the hip circumference. The body shape index was calculated using the following formula (22): ABSI = Wc[cm]/[(BMI) [Kg/m 2 ] (2/3) × (height) [cm] (1/2) ] The body roundness index was calculated using the following formula (23): BRI = 365.2 − 365.5 × √ (1 − (((Wc[cm]/2π ) 2 ) / [(0.5 × height[m])] 2 )) The body adiposity index was calculated using the following formula (24): BAI [% body fat] = (Hc[cm]/ (height [m] ) 1.5 ) – 18 The abdominal volume index was calculated using the following formula (25): AVI= 2Wc[cm] 2 +0.7(Wc[cm]−Hc[cm]) 2 /1000 The conicity index was calculated using the following formula (26): ConI= Wc[cm]/0.109 × √Wt [Kg]/height[m] The participants’ body composition will be assessed using the TANITA BC-418 BIA device, which employs bioelectrical impedance analysis (27). Participants will be required to wear light clothing and be barefoot during the measurement. They will be instructed to position their feet correctly on the foot electrode and hold the hand electrode with both hands, maintaining a 15-degree angle between their torso and upper limb. Upon entering the participants’ index, age, height, and gender into the device, measurements of fat mass (FM), fat-free mass (FFM), and lean body mass (LBM) will be recorded. Nutritional assessments For the purpose of dietary assessment in patients, an appropriate tool for the evaluation of diet (a 3-day food record consisting of two weekdays and one weekend day) will be utilized. A face-to-face interview will be used to complete the 3-day food record at the start, halfway through, and end of the intervention. We will examine the nutritional consumption data provided by the Nutritionist IV software (First Databank, San Bruno, CA, USA) modified for Iranian foods. Participants were also given a list of foods containing probiotics (including yogurt, kefir, buttermilk, cheese, milk, kimchi, kombucha tea, tempeh, apple cider vinegar, balsamic vinegar, soy sauce, sourdough bread, dark chocolate, olives, Parmesan cheese, Pickles, garlic, onions, pickles, feta cheese, smoothies, sauerkraut, mixed pickles, pickled onions, pickled lips, pickled cucumbers, tablets and mouth fresheners containing probiotics) as well as artificial sweeteners containing stevia and mango fruit, and they are asked not to use these products in their diet during the intervention period, and also not to change their diet and physical activity. Additionally, adjustments in dietary intake over the duration of the study will be factored into the statistical analysis to account for potential confounders. GI symptoms questionnaire Gastrointestinal symptom rating scale (GSRS), which has been validated in Iran (28), is used to check the participants gastrointestinal symptoms at the beginning and end of the study. The questionnaire utilized in this study is a hospital assessment tool created by Revicki et al. (29) to assess gastrointestinal symptoms, drawing from clinical experience. The Gastrointestinal Symptom Rating Scale (GSRS) questionnaire comprises 15 questions, each rated on a seven-point Likert scale ranging from no discomfort (score zero) to severe discomfort (score 7). The GSRS tool includes five subscales to evaluate symptoms related to indigestion, constipation, diarrhea, heartburn, and reflux. Higher scores on each subscale indicate greater symptom severity. GSRS score will be calculated as follows: Reflux score: the mean score of items 2 and 3 Abdominal pain score: mean score of items 1, 4, and 5 Indigestion score: mean score of items 6, 7, 8, and 9 Diarrhea score: mean score of items 11, 12, and 14 Constipation score: mean score of items 10, 13, and 15 GSRS score: the mean of all five subgroup scores. Mood status questionnaire In order to check the moods of athletes, Brums moods questionnaire which has been validated in Iran, can be used (30, 31). This questionnaire is the final model of Brums questionnaire with 26 questions to which two subscales with 8 questions have been added, and it measures eight mental states of tension, depression, anger, vitality, fatigue, confusion, peace and happiness. As a result, it consists of 8 subscales (factors). Each scale contains 4 questions. Also, vitality, peace and happiness (12 questions in total) are the positive dimensions of mood states and tension, depression, anger, fatigue and confusion (20 questions in total) are the negative dimensions of mood states in the 32-question Brums questionnaire. Each item in this questionnaire is awarded from zero to five points, so that the option at all is assigned a score of zero and the option completely is assigned a score of five. Finally, the average of 4 items of each factor forms the score. The minimum score of a person in the questionnaire of mood states is equal to zero, and the maximum score is equal to 128 Assessment of muscle strength In adults, handgrip is commonly utilized as a measure of overall muscle strength. This study employs the T.K.K. 5401 handgrip device from Takei Scientific Instruments Co., Ltd to assess muscle strength. During the assessment, participants are instructed to squeeze the clamp with one hand as forcefully as possible for a minimum of 5 seconds and then switch hands at 60-second intervals to prevent fatigue. Each hand undergoes the test three times, and the maximum strength for each hand is documented. Measuring sports performance Athletes’ anaerobic performance will be assessed using the RAST method (32). The test involves athletes running a 35-meter distance six times with 10 seconds of rest between each repetition. Subsequently, the power of each individual repetition will be calculated based on the time recorded for each 35-meter run using a specified formula. Evaluation: according to the following instructions, maximum power, minimum power, average power and fatigue index will be determined: Formula 1: power = Formula 2: maximum power = maximum power between 6 repetitions Formula 3: minimum power = minimum power between 6 repetitions Formula 4: average power = total power of 6 repetitions divided by 6 Formula 5 : Fatigue index = Compliance with intervention To ensure participants’ adherence to the study protocol, regular reminders via messages and phone calls will be implemented on a daily basis, with monitoring every two weeks. In case a daily questionnaire is missed, it will be presumed that the capsule was not taken that day, prompting the participant to consume two capsules the next day. At the end of each month, returned capsules will be tallied to verify intervention compliance. The following formula will be used to calculate the compliance rate, with less than 90% being deemed to be poor compliance. Compliance rate: (Capsules taken / capsules prescribed ( × 100 Statistical analysis Before conducting statistical analyses, all data will be meticulously reviewed for accuracy and completeness. Statistical analysis will be carried out using SPSS software version 27 from SPSS Inc., based in Chicago, IL, USA. Normality of the data will be assessed utilizing the Kolmogorov-Smirnov test. Categorical variables will be compared using Fisher’s exact test and the chi-square test. Continuous variables within groups will be compared using the independent sample t-test and Wilcoxon rank-sum test, while comparisons between groups will utilize the paired sample t-test and Mann-Whitney U test. Normally distributed variables will be represented by mean and standard deviation, whereas non-normally distributed variables will be reported using median and interquartile range (IQR). The analysis of covariance (ANCOVA) test will be utilized to evaluate differences in primary and secondary outcomes between the research groups at the trial’s conclusion and to adjust for potential confounding factors. Statistical significance will be considered at a P value below 0.05. Given the possibility of missing data points in longitudinal studies, a multiple imputation method considering data patterns and the mechanism of missing data will be applied. Analyses for missing data will be conducted on the intention-to-treat (ITT) population, with per-protocol (PP) and sensitivity analyses undertaken as required. Data management and monitoring All data will be securely stored in a centralized database, employing encryption and access controls to maintain confidentiality. The current trial will be overseen by Project Manager N.M. and Research Advisor KJ to ensure data quality and protocol adherence. The Data Monitoring Committee (DMC) will assess the trial procedures and review monitoring outcomes. Adverse events Study participants will be prompted to report any adverse effects (AEs) resulting from the dietary intervention during weekly interactions with investigators. All documented adverse effects will be reported to both the Data Monitoring Committee and the Ethics Committee at the Tehran University of Medical Sciences. Discussion Among athletes, bodybuilders exhibit a higher inclination towards utilizing dietary supplements and hormones in comparison to other athletes (33). Commonly used supplements include whey protein, branched-chain amino acids (BCAA), creatine, and a variety of vitamins (34, 35). Probiotic supplements have gained popularity recently due to their positive impact on the alteration of the gut microbiome (36). Probiotics can influence body composition by promoting a healthy balance of gut microbiota (37). A well-balanced gut microbiome plays a crucial role in nutrient absorption, energy metabolism, and inflammation regulation, potentially affecting body weight and composition (38). Moreover, a healthy gut microbiome contributes to overall well-being, enhancing physical performance (39). Given the promising effects of probiotics on athletes and the limited research on the alteration of gut microbiome in this population, Randomized Controlled Trials (RCTs) are necessary to examine the effects of probiotic supplementation on body composition, gastrointestinal symptoms, mood states, and athletic performance. Recommending a natural component as a means to enhance athletic performance through modifications in an individual’s gut microbiome represents a distinctive feature of the current clinical study. Furthermore, this study could result in cost reduction benefits by addressing the extensive use of supplements and hormones among bodybuilders in Iran and globally. Strength and limitation: This is the first randomized controlled clinical trial that will investigate the effects of probiotic supplementation in unprofessional athletes. The study will assess multiple domains, including performance, body composition, gastrointestinal symptoms, and mood status, allowing for a comprehensive evaluation of probiotic effects. We will examine three of the most commonly used probiotic strains by athletes. Specifically targeting unprofessional bodybuilders, the study may yield insights that are directly relevant to this demographic, helping to improve athletic performance and overall health. However, it is important to note that self-reported measures, such as mood status and gastrointestinal symptoms, can introduce bias and may not accurately reflect true outcomes. Trial status Recruitment for trial ID IRCT20240424061562N1 commenced in August 2024 and remains ongoing as of July 2024. Abbreviations AIS: Australian institute of sports, IOC: International Olympic committee, SCFA: Short-chain fatty acids, HPA: Hypothalamic-pituitary-adrenal axis, VO 2max : Maximal oxygen consumption, CFU: Colony-forming units, BMI: Body mass index, WC: Waist circumference, ABSI: A Body Shape Index, BRI: Body Roundness Index, WHR: waist-to-hip ratio, BAI: body adiposity index, AVI: Abdominal Volume Index, ConI: Conicity Index, WHtR: waist-to-height ratio, FFM: Fat-free mass, FM: Fat mass, LBM: Lean body mass, GSRS: Gastrointestinal symptoms rating scale, BRUMS: Brunel mood scale, IPAQ: International physical activity questionnaire, RAST: Running-based anaerobic sprint test, BCAA: branched-chain amino acids, RCTs: Randomized Controlled Trials Declarations Acknowledgements This study received financial support from Tehran University of Medical Sciences (TUMS), grant number: 72295. The authors express their gratitude to the Tak Gen Zist Pharmaceutical Company in Tehran, Iran. Authors’ contributions The study’s concept was conceived and developed by NM, HM, SSH, SCH, and KDJ. ACH and NM contributed to data collection. NM drafted initial versions of the manuscript. Statistical analysis advice was provided by SSH and HM. KDJ contributed to the final editing of the manuscript. All authors have reviewed and approved the final draft. Funding The trial received funding from Tehran University of Medical Sciences with grant number 72295. The funder had no role in study design, data collection, analysis, interpretation, manuscript writing, or publication decision. Availability of data and materials The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Ethics approval The trial received ethical approval from the Medical Ethics Committee at Tehran University of Medical Sciences, ethical code: IR.TUMS.MEDICINE.REC.1403.157. Consent for publication Specific publication details have been withheld. 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A new index of abdominal adiposity as an indicator of risk for cardiovascular disease. A cross-population study. International journal of obesity and related metabolic disorders: journal of the International Association for the Study of Obesity. 1993;17(2):77-82. Valdez R. A simple model-based index of abdominal adiposity. Journal of clinical epidemiology. 1991;44(9):955-6. Kelly JS, Metcalfe J. Validity and Reliability of Body Composition Analysis Using the Tanita BC418-MA. Journal of Exercise Physiology Online. 2012;15(6). Mazaheri M, Khoshouei MS. Comparison between psychometric characteristics of persian version of the gastrointestinal symptoms rating scale in functional gastrointestinal disorders and normal groups. 2012. Revicki DA, Wood M, Wiklund I, Crawley J. Reliability and validity of the Gastrointestinal Symptom Rating Scale in patients with gastroesophageal reflux disease. Quality of life research. 1997;7:75-83. FAROKHI A, MOTESHAREIE E, ZEIDABADY R. Validity and reliability of Persian version of Brunel mood scale 32 items. 2013. Bagherpour T, Hashim HA, Saha S. Factorial Validity and Internal Consistency of Iranian Adapted Brunel Mood Scale among Iranian Young Female Taekwondo Athletes. J Phys Act Sport Exerc. 2013;1(1):74-7. Abbasian S, Golzar S, Onvani V, Sargazi L. The predict of RAST Test from WANT test in Elite Athletes. Research Journal of Recent Sciences. 2012;2277:2502. Sánchez-Oliver AJ, Grimaldi-Puyana M, Domínguez R. Evaluation and behavior of spanish bodybuilders: Doping and sports supplements. Biomolecules. 2019;9(4):122. Khudhur AF. Whey Protein Supplements between Bodybuilders. Health, Risk of Diseases and Myth: A Narrative Review. 2010;2024:49-55. Ghorashi A, Moghaddam RH, Mehrpooya M, Rangchian M. Pattern and Correctness of Male Bodybuilders' Supplements and Medicines Consumption: An Evaluation by Pharmacist. J Pharmaceutics and Pharmacology Research. 2022;5(3). Gryaznova M, Smirnova Y, Burakova I, Syromyatnikov M, Chizhkov P, Popov E, et al. Changes in the human gut microbiome caused by the short-term impact of lactic acid bacteria consumption in healthy people. Probiotics and Antimicrobial Proteins. 2024;16(4):1240-50. Hassan NE, El-Masry SA, El Shebini SM, Ahmed NH, Mehanna NS, Abdel Wahed MM, et al. Effect of weight loss program using prebiotics and probiotics on body composition, physique, and metabolic products: longitudinal intervention study. Scientific Reports. 2024;14(1):10960. Ross FC, Patangia D, Grimaud G, Lavelle A, Dempsey EM, Ross RP, et al. The interplay between diet and the gut microbiome: implications for health and disease. Nature Reviews Microbiology. 2024:1-16. Varghese S, Rao S, Khattak A, Zamir F, Chaari A. Physical Exercise and the Gut Microbiome: A Bidirectional Relationship Influencing Health and Performance. Nutrients. 2024;16(21):3663. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files supplementaryfileSPIRIT2013Checklist.docx appendix1.docx table1.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6712303","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":474452706,"identity":"4c06ef66-f210-49b4-b153-ba9076e2654b","order_by":0,"name":"Nastaran Mahmoudi Shirkoohi","email":"","orcid":"","institution":"Tehran University of medical science","correspondingAuthor":false,"prefix":"","firstName":"Nastaran","middleName":"Mahmoudi","lastName":"Shirkoohi","suffix":""},{"id":474452707,"identity":"e14d7869-4319-4b46-914f-94261fcf8add","order_by":1,"name":"Alireza Chegol","email":"","orcid":"","institution":"Tehran University of medical 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04:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6712303/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6712303/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85202363,"identity":"33ba61fc-8e3b-4781-9822-0b11a76e1058","added_by":"auto","created_at":"2025-06-23 10:45:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":416765,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy flowchart.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6712303/v1/784cdb03ada5af704f1ea21b.jpg"},{"id":85203077,"identity":"473ac6b6-530b-436c-8eac-3ab471b31b1f","added_by":"auto","created_at":"2025-06-23 10:53:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":598219,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSPIRIT diagram of recommended content for the schedule of enrolment, interventions, and assessments.*\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6712303/v1/df73538b7717ece4dd67ca3b.jpg"},{"id":89251869,"identity":"225b1004-38c8-449f-9978-b1c92bf237b4","added_by":"auto","created_at":"2025-08-18 04:01:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1966594,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6712303/v1/e69c4e90-6d4a-4f17-a8ca-94047ef1daab.pdf"},{"id":85202365,"identity":"99f254e2-7206-4cb1-843a-395a7086300b","added_by":"auto","created_at":"2025-06-23 10:45:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":51077,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfileSPIRIT2013Checklist.docx","url":"https://assets-eu.researchsquare.com/files/rs-6712303/v1/257bd5f58a69d22cc4d3cce7.docx"},{"id":85203079,"identity":"43a1be0d-b06f-4a7d-9125-f59cc16fb26f","added_by":"auto","created_at":"2025-06-23 10:53:34","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19499,"visible":true,"origin":"","legend":"","description":"","filename":"appendix1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6712303/v1/dd8122fc71879b39ff0a792f.docx"},{"id":85202367,"identity":"2987d345-7559-4b35-9f7d-1cfedb8e5ce3","added_by":"auto","created_at":"2025-06-23 10:45:34","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":53248,"visible":true,"origin":"","legend":"","description":"","filename":"table1.doc","url":"https://assets-eu.researchsquare.com/files/rs-6712303/v1/9caf5289515047087d769606.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Effects of Probiotic Supplementation on Performance, Body composition, Gastro- intestinal Symptoms and Mood Status in Unprofessional Athletes: Study Protocol for a Randomized Double-blind Clinical Trial","fulltext":[{"header":"What is already known about this topic? ","content":"\u003cp\u003eProbiotic supplementation is known to improve gut health by enhancing microbiota diversity, which can alleviate gastrointestinal symptoms common in athletes. Additionally, there is evidence suggesting that probiotics may enhance exercise performance through better nutrient absorption and digestion, support immune function to reduce illness risk, and positively impact mood and mental well-being, potentially alleviating anxiety and depression. Some strains may also assist in body composition management, yet research findings vary due to factors like strain specificity and individual differences. Most existing studies focus on professional athletes, highlighting a gap in understanding the effects of probiotics on unprofessional athletes, which the current study aims to address.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhat does this article add?\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article adds significant value by focusing on unprofessional athletes, a demographic often neglected in probiotic research. It employs a rigorous randomized double-blind clinical trial design to provide a comprehensive evaluation of probiotic supplementation, examining its effects on performance, body composition, gastrointestinal symptoms, and mood status. By specifically investigating three commonly used probiotic strains among athletes, the study aims to identify which formulations may be most beneficial. The findings could lead to practical dietary recommendations, helping unprofessional athletes optimize their health and performance. Overall, this research enhances the understanding of probiotics in athletic contexts.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003e\u003cstrong\u003eBackground and rationale\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAthletes are always looking for ways to improve their performance. The success of an athlete is dependent on characteristics such as genetic, physiological, and psychological characteristics, and on the other hand, it is dependent on targeted and scientific planning in the framework of regular and organized training and nutrition (1). Fatigue, mood disturbances, and gastrointestinal distress are the most important obstacles for athletes to achieve better performance (2). Therefore, due to the desire of athletes to achieve their athletic goals quickly, they often resort to supplements, energy boosters, and occasionally even misuse medications (3). The consumption of various supplements is observed not only among professional athletes, but also among beginner athletes and non-athletes (4). Additionally, research indicates a high prevalence of supplement usage among Iranian athletes, standing at 64.8% (5). But one of the supplements that is less commonly used by athletes is probiotics.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eProbiotics are often overlooked supplements that are crucial for both health and enhancing athletes\u0026rsquo; performance (6). According to the classification of sports supplements by the sports nutrition department of the Australian Institute of sports (AIS), probiotics are class A (first) supplements, so it seems that this supplement can have an increasing effect on improving sports performance (7). The International Olympic Committee (IOC) introduces probiotics as useful living microorganisms for gut health and immune system modulation (8). Studies have highlighted the beneficial impact of probiotics on enhancing the immune system post intense exercise, alleviating digestive issues through microbiota modulation and SCFA production, mitigating respiratory symptoms, and lowering the susceptibility to respiratory infections (9, 10). In addition, intestinal dysbiosis activates the hypothalamic-pituitary-adrenal axis (HPA), leading to depression and anxiety. Probiotics can improve gut dysbiosis by suppressing this overactivated axis, which decreases serotonin and increases inflammatory cytokines (11-13). Numerous researchers have highlighted the advantages of probiotic use in enhancing athletic performance (14). Mazur-Kurach et al. in 2022 (15) investigated the role of probiotics in the sports performance of road cyclists. The findings showed that probiotics increased maximal oxygen consumption (VO\u003csub\u003e2max\u003c/sub\u003e), training time to exhaustion, load on the ergometer, and the degree of heart rate reduction. Despite its impact on athletes\u0026rsquo; aerobic capacity, probiotics did not show any noticeable effects on anaerobic capacity or body composition. In an investigation conducted by Salleh et al (16) among 30 badminton players, the results showed 6 weeks of supplementation with probiotics significantly increased the aerobic capacity of athletes. Also, the consumption of this supplement led to a reduction in the level of stress and anxiety and improved mood in athletes. In a study by Schreiber et al. (17) on elite cyclists over a 90-day period, probiotics were examined for their impact on sports performance, inflammatory markers, and gastrointestinal symptoms. The study results revealed no significant disparities in VO\u003csub\u003e2max\u003c/sub\u003e, maximum heart rate, or time to fatigue between the intervention and placebo groups. However, the findings did demonstrate that probiotic supplementation led to improvements in digestive symptoms among the participants. Probiotic treatment might affect physiological responses to exercise greater among unprofessional athlete, as well as immunomodulatory and gastrointestinal benefits that could improve performance and benefit athletes (18).\u0026nbsp;Thus, the goals and hypotheses of this double-blind, randomized placebo-controlled clinical trial will be as follows.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study\u0026rsquo;s main goal is to evaluate the ergogenic effects of probiotic supplementation on anaerobic performance in unprofessional athletes. Secondary objectives include assessment of body composition, gastro-intestinal symptoms, mood status, and muscle strength.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA randomized, parallel, double-blind and placebo-controlled clinical trial will be implemented.\u003c/p\u003e"},{"header":"Methods: participants, interventions, and outcomes","content":"\u003cp\u003e\u003cstrong\u003eStudy setting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study is a placebo-controlled, randomized, double-blind clinical trial that explore the impacts of probiotic use in non-professional athletes. This study was conducted according to the Helsinki Declaration and written informed consent was obtained from all participants. The research has obtained approval from the Medical Ethics Committee at Tehran University of Medical Sciences in Iran ethical code: IR.TUMS.MEDICINE.REC.1403.157 and is already registered in the Iranian Registry of Clinical Trials under the ID IRCT20240424061562N1. The study will be presented following the CONSORT 2010 guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEligibility criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants were included in the current trial if met our inclusion criteria: 1) healthy adults aged 20\u0026ndash;40 years old who have an average of 6\u0026ndash;10 hours of training per week; 2) a history of at least 3 months of bodybuilding training; 3) athletes who consent to participate in this study by signing a formal informed consent form.\u003c/p\u003e\n\u003cp\u003eNon-inclusion criteria were as follows: 1) professional athletes who practice for more than 6 hours a week; 2) pregnancy and lactating; 3) premature menopause; 4) Various medical conditions can impact individuals\u0026rsquo; capacity to participate in or partake in physical activity; 5) frequent intake of probiotic products, for example dietary fibers such as inulin and galacto-oligosaccharides, regularly 1 month before and during the study; 6) smokers who are currently in the habit, suspected or confirmed history of alcohol or\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003edrug misuse; 7) consumption of food and sports supplements at least four to six weeks prior to and during the intervention; 8) adhering special diets; 9) taken any antibiotics during the course of the treatment or in the past four weeks; 10) having gastrointestinal diseases, i.e., food allergies, celiac, irritable bowel disease, Whitin the last two months; 11) engaging in sports other than bodybuilding; 12) having acute gastrointestinal symptoms up to two months before the study.\u003c/p\u003e\n\u003cp\u003eExclusion criteria: 1) Allergy to studied agents 2) Noncompliance in consumption of the supplements (\u0026lt;90%) 3) taking antibiotics during the study 4) pregnancy during study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt baseline, the trial\u0026rsquo;s goal will be explained to the legal guardian by the principal investigator (N.M), who will obtain their written informed consent upon enrollment. Also, they will be assured that their information will remain confidential \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eAppendix1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterventions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty-six eligible participants will be randomly assigned to either the placebo group (n = 18) or the intervention group (n = 18). Participants in both groups will be administered probiotic capsules containing Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei (2 billion colony-forming units), along with identical placebo capsules containing 100 mg of maltodextrin daily with a meal for a duration of 12 weeks. The Tak Gen Zist Pharmaceutical Company, Tehran, Iran, will manufacture the intervention and control boxes. They will be equivalent in size, taste, smell, and package (tagged 1 and 2), allowing for allocation concealment. Two separate microbiologists verified microbiological and purity tests. Any clinical adverse event (AE) linked to the intervention, laboratory abnormalities, or other medical conditions (such as pregnancy) that would not benefit the participant\u0026rsquo;s well-being will be grounds for discontinuing the intervention in the study.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eAll supplements and tests in this program will be provided to athlete free of charge, and during the three months they patriciate in this study, they will receive free diet by nutritionist. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOutcomes:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary outcome assesses alterations in RAST (Running-based anaerobic sprint test) performance metrics, such as minimum, average, peak power, and fatigue index, in the probiotic and placebo groups after a 12-week intervention compared to baseline.\u003c/p\u003e\n\u003cp\u003eSecondary outcomes, involving a comparison of the probiotic and placebo groups while accounting for their respective baseline values, include the following: 1) changes in body composition and anthropometric indices (weight, body mass index (BMI), waist circumference (WC), fat-free mass (FFM), fat mass (FM), lean body mass (LBM), a body shape index (ABSI), body roundness index (BRI), waist-to-hip ratio (WHR),\u0026nbsp;body adiposity index (BAI), abdominal volume index (AVI), conicity Index (ConI), and waist-to-height ratio (WHtR)) from baseline; 2) gastrointestinal symptoms that will be evaluated through the Gastrointestinal Symptoms Rating Scale (GSRS) questionnaire at the beginning and end of the study; 3) mood status that will be evaluated through Brunel Mood Scale (BRUMS) questionnaire at the beginning and end of the study; 4) changes in muscle strength\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticipant timeline\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe timeline of the trial and study flow chart of enrollment, allocation, intervention, and assessment are presented in \u003cstrong\u003eTable 1\u003c/strong\u003e and \u003cstrong\u003eFig. 1\u003c/strong\u003e, respectively. Initially, all participants who consent will undergo a 2-week run-in phase, during which three 24-hour food recalls (two on working days and one on a weekend) will be conducted to collect dietary information. Demographic details, physical activity levels, medical history, medication usage, and socioeconomic status data will be collected through a questionnaire and face-to-face interviews. Following the run-in phase, assessments will be made on body composition, GI symptoms, mood status, and performance. Subsequently, participants will be categorized by gender (male/female) into blocks and then randomly assigned to either the intervention or control groups. After 6 weeks, participants will undergo another round of 24-hour food recalls. Finally, following the conclusion of the study, all outcomes will be reassessed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample size\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sample size was determined to be 16 for each group using a two-sided t-test, considering a type I error of 5% (\u0026alpha; = 0.05), an 80% power, and sport performance as the primary outcome (19). To accommodate an anticipated 10% dropout rate during the study, the final sample size was increased to 18 participants per group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecruitment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe recruitment for this study is set to commence in August 2024 and is anticipated to conclude in February 2024. Participants are invited to participate in this project based on the advertisement in social networks, sports clubs and sports medicine clinics. If they wish to cooperate, they will be informed about the implementation of the project in general, then we will provide the necessary explanations about the probiotic supplement and the purpose of the study to the people, then informed written consent will be obtained from the participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRandomization, sequence generation, concealment, and blinding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipant randomization to the intervention/placebo sequence will be carried out by a local pharmacist utilizing permuted-block randomization stratified by gender (block sizes 4, 6, 6) through the designated website: https://www.sealedenvelope.com/simple-randomiser/v1/lists [Accessed 2 Aug 2024]. The experimental and placebo nutritional supplements will be dispensed to participants by the pharmacist in the specified sequence. The pharmacist\u0026rsquo;s role will solely involve providing the supplements, without any direct interaction with the study participants or involvement in data collection or processing. Opaque and sealed containers containing dietary supplements will be distributed monthly and monitored for compliance by weighing. External pharmaceutical packaging services will handle the off-site packaging of supplements. Each package will bear a participant\u0026rsquo;s study identification number and be labeled as Supplement 1 or Supplement 2 based on the intervention phase to ensure treatment concealment from volunteers and investigators. Data collectors and data administrators will be blinded to the group assignment. Unblinding will only occur after the completion of statistical analysis of the data or if serious adverse events occur, suspicious unintended serious adverse reactions, it can be uncovered after asking the leading researcher. Furthermore, supplement delivery to participants will occur on the 1st, 30th, and 60th days according to the pre-determined allocation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection and management\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of physical activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study will involve assessing physical activity levels using the abbreviated version of the International Physical Activity Questionnaire (IPAQ)(20). Measurements will be reported in MET-min per week on the IPAQ form. A scoring method based on IPAQ will classify participants into low, moderate, or high activity level groups (21). Throughout the study, adjustments in physical activity levels will be considered during statistical analysis to address potential confounding factors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnthropometric and body composition assessments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the trial, height and body weight will be measured using the Seca scale and stadiometer with precision to the nearest 0.1 cm and 0.1 kg, respectively. Participants will be required to be barefoot and dressed comfortably for these measurements. Body Mass Index (BMI) will be calculated by dividing weight in kilograms by the square of height in meters. Waist circumference (WC) will be determined by measuring the greatest gluteal muscle, the middle of the lowest rib, and the highest point of the iliac crest. Hip circumference will be measured at the widest point over the buttocks with a measuring tape accurate to 0.1 cm. The Waist-to-Hip Ratio (WHR) can be calculated by dividing the waist circumference by the hip circumference.\u003c/p\u003e\n\u003cp\u003eThe body shape index was calculated using the following formula (22):\u003c/p\u003e\n\u003cp\u003eABSI = Wc[cm]/[(BMI) [Kg/m\u003csup\u003e2\u003c/sup\u003e] \u003csup\u003e(2/3)\u003c/sup\u003e \u0026times; (height) [cm] \u003csup\u003e(1/2)\u003c/sup\u003e]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe body roundness index was calculated using the following formula (23):\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBRI = 365.2 \u0026minus; 365.5 \u0026times; \u0026radic; (1 \u0026minus; (((Wc[cm]/2\u0026pi; )\u003csup\u003e2\u003c/sup\u003e) / [(0.5 \u0026times; height[m])] \u003csup\u003e2\u003c/sup\u003e))\u003c/p\u003e\n\u003cp\u003eThe body adiposity index was calculated using the following formula (24):\u003c/p\u003e\n\u003cp\u003eBAI [% body fat] = (Hc[cm]/ (height [m] )\u003csup\u003e1.5\u003c/sup\u003e) \u0026ndash; 18\u003c/p\u003e\n\u003cp\u003eThe abdominal volume index was calculated using the following formula (25):\u003c/p\u003e\n\u003cp\u003eAVI= 2Wc[cm]\u003csup\u003e2\u003c/sup\u003e+0.7(Wc[cm]\u0026minus;Hc[cm])\u003csup\u003e2\u003c/sup\u003e/1000\u003c/p\u003e\n\u003cp\u003eThe conicity index was calculated using the following formula (26):\u003c/p\u003e\n\u003cp\u003eConI= Wc[cm]/0.109 \u0026times; \u0026radic;Wt [Kg]/height[m]\u003c/p\u003e\n\u003cp\u003eThe participants\u0026rsquo; body composition will be assessed using the TANITA BC-418 BIA device, which employs bioelectrical impedance analysis (27).\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eParticipants will be required to wear light clothing and be barefoot during the measurement. They will be instructed to position their feet correctly on the foot electrode and hold the hand electrode with both hands, maintaining a 15-degree angle between their torso and upper limb. Upon entering the participants\u0026rsquo; index, age, height, and gender into the device, measurements of fat mass (FM), fat-free mass (FFM), and lean body mass (LBM) will be recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNutritional assessments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the purpose of dietary assessment in patients, an appropriate tool for the evaluation of diet (a 3-day food record consisting of two weekdays and one weekend day) will be utilized. A face-to-face interview will be used to complete the 3-day food record at the start, halfway through, and end of the intervention. We will examine the nutritional consumption data provided by the Nutritionist IV software (First Databank, San Bruno, CA, USA) modified for Iranian foods. Participants were also given a list of foods containing probiotics (including yogurt, kefir, buttermilk, cheese, milk, kimchi, kombucha tea, tempeh, apple cider vinegar, balsamic vinegar, soy sauce, sourdough bread, dark chocolate, olives, Parmesan cheese, Pickles, garlic, onions, pickles, feta cheese, smoothies, sauerkraut, mixed pickles, pickled onions, pickled lips, pickled cucumbers, tablets and mouth fresheners containing probiotics) as well as artificial sweeteners containing stevia and mango fruit, and they are asked not to use these products in their diet during the intervention period, and also not to change their diet and physical activity. Additionally, adjustments in dietary intake over the duration of the study will be factored into the statistical analysis to account for potential confounders.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGI symptoms questionnaire\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGastrointestinal symptom rating scale (GSRS), which has been validated in Iran (28), is used to check the participants gastrointestinal symptoms at the beginning and end of the study. The questionnaire utilized in this study is a hospital assessment tool created by Revicki et al. (29) to assess gastrointestinal symptoms, drawing from clinical experience. The Gastrointestinal Symptom Rating Scale (GSRS) questionnaire comprises 15 questions, each rated on a seven-point Likert scale ranging from no discomfort (score zero) to severe discomfort (score 7). The GSRS tool includes five subscales to evaluate symptoms related to indigestion, constipation, diarrhea, heartburn, and reflux. Higher scores on each subscale indicate greater symptom severity.\u003c/p\u003e\n\u003cp\u003eGSRS score will be calculated as follows:\u003c/p\u003e\n\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003eReflux score: the mean score of items 2 and 3\u003c/li\u003e\n \u003cli\u003eAbdominal pain score: mean score of items 1, 4, and 5\u003c/li\u003e\n \u003cli\u003eIndigestion score: mean score of items 6, 7, 8, and 9\u003c/li\u003e\n \u003cli\u003eDiarrhea score: mean score of items 11, 12, and 14\u003c/li\u003e\n \u003cli\u003eConstipation score: mean score of items 10, 13, and 15\u003c/li\u003e\n \u003cli\u003eGSRS score: the mean of all five subgroup scores.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eMood status questionnaire\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to check the moods of athletes, Brums moods questionnaire which has been validated in Iran, can be used (30, 31). This questionnaire is the final model of Brums questionnaire with 26 questions to which two subscales with 8 questions have been added, and it measures eight mental states of tension, depression, anger, vitality, fatigue, confusion, peace and happiness. As a result, it consists of 8 subscales (factors). Each scale contains 4 questions. Also, vitality, peace and happiness (12 questions in total) are the positive dimensions of mood states and tension, depression, anger, fatigue and confusion (20 questions in total) are the negative dimensions of mood states in the 32-question Brums questionnaire. Each item in this questionnaire is awarded from zero to five points, so that the option at all is assigned a score of zero and the option completely is assigned a score of five. Finally, the average of 4 items of each factor forms the score. The minimum score of a person in the questionnaire of mood states is equal to zero, and the maximum score is equal to 128\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of muscle strength\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn adults, handgrip is commonly utilized as a measure of overall muscle strength. This study employs the T.K.K. 5401 handgrip device from Takei Scientific Instruments Co., Ltd to assess muscle strength. During the assessment, participants are instructed to squeeze the clamp with one hand as forcefully as possible for a minimum of 5 seconds and then switch hands at 60-second intervals to prevent fatigue. Each hand undergoes the test three times, and the maximum strength for each hand is documented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasuring sports performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAthletes\u0026rsquo; anaerobic performance will be assessed using the RAST method (32). The test involves athletes running a 35-meter distance six times with 10 seconds of rest between each repetition. Subsequently, the power of each individual repetition will be calculated based on the time recorded for each 35-meter run using a specified formula. Evaluation: according to the following instructions, maximum power, minimum power, average power and fatigue index will be determined:\u003c/p\u003e\n\u003cp\u003eFormula 1: power =\u0026nbsp;\u003cimg width=\"112\" height=\"31\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAHAAAAAfCAMAAAAInLn2AAAAAXNSR0IArs4c6QAAANJQTFRFAAAAAAAAAAA6AABmADo6ADpmADqQAGaQAGa2OgAAOgA6OgBmOjo6OjpmOjqQOmaQOma2OpC2OpDbZgAAZgA6ZgBmZjoAZjo6ZjpmZjqQZmZmZmaQZma2ZpDbZrbbZrb/kDoAkDo6kDpmkGY6kGZmkGaQkJA6kJBmkJDbkLb/kNv/tmYAtmY6tmZmtpA6tpC2trbbtrb/ttuQttu2ttvbttv/tv//25A625Bm25C227Zm27aQ27a229uQ29vb2/+22////7Zm/9uQ/9u2//+2///b6aMZTgAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAADsQAAA7EAZUrDhsAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAACdElEQVRIS+1WbXfSMBROOql0UwbtqFM3CmP40uLLnFCsU9M2+f9/yefeBKYe7A5b9cPO7oE0ubnvN+0TIR7ozwrU54sdi1Kc7qjwm7h+rnZWr/oNKmaSrnczbzO9VsgTO89lIn6RbQ5iToaKQB42B+uS0cdLIcwZBkTKT6LNhBYs0kB5COWeqiNEm+0tRZboqCvMXHo8wWYsn4SiOpxKjIGU4NXHy+qlEnpIps1UPvIVy5aB9D+xCJier+q4WwS+ohXVgGxCiIy6IPVRWgWJWKVmMvo+TwQHs49VKvK9BUkSB1Q/ewpf7NCMB4q4+EPOzHjBzK64+rY/QmlotbG5driidkDj3etQv6FMvIESVO4sZMNZQj/mUNbs0Ja2Bzm3U8V9q0RMEofTss8r8KxNF/PqLRvKXpyWj2eKoqhnSg8vL6BnpqjYcGnOPn/E4wt0qKTUKTo0pa+KA9q5vFil1BAScUwOc/ShQHFPnE0XM/IrKUVEVB1wnWRnIXSE+hey8wrN6yk97iz1mLpgzu2hEXSSdOyNaAeymZQIjUTWzKNUvPdSWMORtDZZj88CurqFqH011P5C9az1Fz+X0rvTx6HxFbn/m+jkf6X7X9GHDG0FGlH+boC+tcTXKL8VcunLZSbyJnjdoXuM8g0gC7BATAC6HWw2iVpIIpAlyK3i7irwiwDA7eDVoc/XE0KkNsiiPOMwBkbYaPAD4GUh2wK6jtr7/lqHjMM0EJ71FP0cvDpA5xtLK8QlZZB18EyJ5uFVwZBt84Z/M27LIaO8BVlALl0E6MLjjRy8ct5op8RhbYluuPw23nNvF0Ijyv+DF/92UW7X+glqOWlwtLFiqAAAAABJRU5ErkJggg==\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eFormula 2: maximum power = maximum power between 6 repetitions\u003c/p\u003e\n\u003cp\u003eFormula 3: minimum power = minimum power between 6 repetitions\u003c/p\u003e\n\u003cp\u003eFormula 4: average power = total power of 6 repetitions divided by 6\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFormula 5 : Fatigue index =\u0026nbsp;\u003cimg width=\"197\" height=\"30\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ensure participants\u0026rsquo; adherence to the study protocol, regular reminders via messages and phone calls will be implemented on a daily basis, with monitoring every two weeks. In case a daily questionnaire is missed, it will be presumed that the capsule was not taken that day, prompting the participant to consume two capsules the next day. At the end of each month, returned capsules will be tallied to verify intervention compliance. The following formula will be used to calculate the compliance rate, with less than 90% being deemed to be poor compliance. Compliance rate: (Capsules taken\u003cspan dir=\"RTL\"\u003e/\u0026nbsp;\u003c/span\u003ecapsules prescribed \u003cspan dir=\"RTL\"\u003e(\u003c/span\u003e\u0026times; 100\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore conducting statistical analyses, all data will be meticulously reviewed for accuracy and completeness. Statistical analysis will be carried out using SPSS software version 27 from SPSS Inc., based in Chicago, IL, USA. Normality of the data will be assessed utilizing the Kolmogorov-Smirnov test. Categorical variables will be compared using Fisher\u0026rsquo;s exact test and the chi-square test. Continuous variables within groups will be compared using the independent sample t-test and Wilcoxon rank-sum test, while comparisons between groups will utilize the paired sample t-test and Mann-Whitney U test. Normally distributed variables will be represented by mean and standard deviation, whereas non-normally distributed variables will be reported using median and interquartile range (IQR). The analysis of covariance (ANCOVA) test will be utilized to evaluate differences in primary and secondary outcomes between the research groups at the trial\u0026rsquo;s conclusion and to adjust for potential confounding factors. Statistical significance will be considered at a P value below 0.05. Given the possibility of missing data points in longitudinal studies, a multiple imputation method considering data patterns and the mechanism of missing data will be applied. Analyses for missing data will be conducted on the intention-to-treat (ITT) population, with per-protocol (PP) and sensitivity analyses undertaken as required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData management and monitoring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data will be securely stored in a centralized database, employing encryption and access controls to maintain confidentiality.\u0026nbsp;The current trial will be overseen by Project Manager N.M. and Research Advisor KJ to ensure data quality and protocol adherence. The Data Monitoring Committee (DMC) will assess the trial procedures and review monitoring outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdverse events\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy participants will be prompted to report any adverse effects (AEs) resulting from the dietary intervention during weekly interactions with investigators. All documented adverse effects will be reported to both the Data Monitoring Committee and the Ethics Committee at the Tehran University of Medical Sciences.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAmong athletes, bodybuilders exhibit a higher inclination towards utilizing dietary supplements and hormones in comparison to other athletes (33).\u0026nbsp;Commonly used supplements include whey protein, branched-chain amino acids (BCAA), creatine, and a variety of vitamins (34, 35). Probiotic supplements have gained popularity recently due to their positive impact on the alteration of the gut microbiome (36). Probiotics can influence body composition by promoting a healthy balance of gut microbiota (37). A well-balanced gut microbiome plays a crucial role in nutrient absorption, energy metabolism, and inflammation regulation, potentially affecting body weight and composition (38). \u0026nbsp;Moreover, a healthy gut microbiome contributes to overall well-being, enhancing physical performance (39). Given the promising effects of probiotics on athletes and the limited research on the alteration of gut microbiome in this population, Randomized Controlled Trials (RCTs) are necessary to examine the effects of probiotic supplementation on body composition, gastrointestinal symptoms, mood states, and athletic performance. Recommending a natural component as a means to enhance athletic performance through modifications in an individual\u0026rsquo;s gut microbiome represents a distinctive feature of the current clinical study. Furthermore, this study could result in cost reduction benefits by addressing the extensive use of supplements and hormones among bodybuilders in Iran and globally.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrength and limitation:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is the first randomized controlled clinical trial that will investigate the effects of probiotic supplementation in unprofessional athletes. The study will assess multiple domains, including performance, body composition, gastrointestinal symptoms, and mood status, allowing for a comprehensive evaluation of probiotic effects. We will examine three of the most commonly used probiotic strains by athletes. Specifically targeting unprofessional bodybuilders, the study may yield insights that are directly relevant to this demographic, helping to improve athletic performance and overall health. However, it is important to note that self-reported measures, such as mood status and gastrointestinal symptoms, can introduce bias and may not accurately reflect true outcomes.\u003c/p\u003e"},{"header":"Trial status","content":"\u003cp\u003eRecruitment for trial ID IRCT20240424061562N1 commenced in August 2024 and remains ongoing as of July 2024.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAIS: Australian institute of sports, IOC: International Olympic committee, SCFA: Short-chain fatty acids, HPA: Hypothalamic-pituitary-adrenal axis, VO\u003csub\u003e2max\u003c/sub\u003e: Maximal oxygen consumption, CFU: Colony-forming units, BMI: Body mass index, WC: Waist circumference, ABSI: A Body Shape Index, BRI: Body Roundness Index, WHR: waist-to-hip ratio, BAI: body adiposity index, AVI: Abdominal Volume Index, ConI: Conicity Index, WHtR: waist-to-height ratio, FFM: Fat-free mass, FM: Fat mass, LBM: Lean body mass, GSRS: Gastrointestinal symptoms rating scale, BRUMS: Brunel mood scale, IPAQ: International physical activity questionnaire, RAST: Running-based anaerobic sprint test, BCAA: branched-chain amino acids, RCTs: Randomized Controlled Trials\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received financial support from Tehran University of Medical Sciences (TUMS), grant number: 72295. The authors express their gratitude to the Tak Gen Zist Pharmaceutical Company in Tehran, Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study\u0026rsquo;s concept was conceived and developed by NM, HM, SSH, SCH, and KDJ. ACH and NM contributed to data collection. NM drafted initial versions of the manuscript. Statistical analysis advice was provided by SSH and HM. KDJ contributed to the final editing of the manuscript. All authors have reviewed and approved the final draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trial received funding from Tehran University of Medical Sciences with grant number 72295. The funder had no role in study design, data collection, analysis, interpretation, manuscript writing, or publication decision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trial received ethical approval from the Medical Ethics Committee at Tehran University of Medical Sciences, ethical code: IR.TUMS.MEDICINE.REC.1403.157.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecific publication details have been withheld. Editors and reviewers have endorsed the material supporting the authors\u0026rsquo; claims.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHakimi M, Sheikholeslami-Vatani D, Ali-Mohammadi M. Effect of concurrent training with ingested of L-carnitine supplementation on hormonal changes, lipid profile and body composition in obese men. 2015.\u003c/li\u003e\n\u003cli\u003eClark A, Mach N. Exercise-induced stress behavior, gut-microbiota-brain axis and diet: a systematic review for athletes. Journal of the International Society of Sports Nutrition. 2016;13:1-21.\u003c/li\u003e\n\u003cli\u003eTymowski G, Somerville C. The use of performance-enhancing substances by New Brunswick students: Pilot study report. 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Nutrients. 2024;16(21):3663.\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Probiotic supplementation, Gastrointestinal symptoms, Mood states, Exercise performance, Unprofessional Athlete","lastPublishedDoi":"10.21203/rs.3.rs-6712303/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6712303/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Probiotic supplementation has emerged as a promising strategy in sport nutrition, with potential benefits for various aspects of athletes' health and performance. The study aims to explore the impact of probiotic supplementation on performance, body composition, gastrointestinal symptoms, and mood status in non-elite athletes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods/Design:\u003c/strong\u003e In this double-blind, randomized controlled clinical trial, 36 non-elite athletes were randomly assigned to receive a probiotic supplement (containing 2×10\u003csup\u003e9\u003c/sup\u003e colony-forming units Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei) or identical placebo once a day for 12 weeks. The body composition, muscle strength, and performance will be measured at baseline and following supplementation. The sociodemographic, physical activity and dietary intake questionnaires will be collected for each subject. Additional questionnaires will inquire about GI symptoms, and mood status at baseline and following treatment. The evaluation of primary and secondary outcomes will involve comparing results among intervention groups, considering baseline values.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion:\u003c/strong\u003e By promoting a healthy balance of gut microbiota, probiotics may offer a natural and effective way to improve overall well-being and enhance various aspects of performance. This trial’s findings may be incorporated in to evidence-based guidelines that address the alternation of gut microbiota to enhance athletic performance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and dissemination:\u003c/strong\u003e There are so far no reported adverse effects associated with the use of probiotic. The research has obtained approval from the Medical Ethics Committee at Tehran University of Medical Sciences in Iran ethical code: IR.TUMS.MEDICINE.REC.1403.157\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration:\u003c/strong\u003e IRCT20240424061562N1, registered on 30/7/2024. https://www.irct.ir/trial/77722.\u003c/p\u003e","manuscriptTitle":"The Effects of Probiotic Supplementation on Performance, Body composition, Gastro- intestinal Symptoms and Mood Status in Unprofessional Athletes: Study Protocol for a Randomized Double-blind Clinical Trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-23 10:45:29","doi":"10.21203/rs.3.rs-6712303/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ecc23262-2d7c-4032-bff9-ce2d113fcb67","owner":[],"postedDate":"June 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50386650,"name":"Health sciences/Health care/Nutrition"},{"id":50386651,"name":"Biological sciences/Psychology"}],"tags":[],"updatedAt":"2025-08-18T03:53:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-23 10:45:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6712303","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6712303","identity":"rs-6712303","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}