Effects of 3-month Probiotic Mix Supplementation (L. Helveticus R-0052, B. Longum R-0175) on Gut Microbiota and Metabolome, Endocannabinoid and Immune Systems Activation, Along With Symptoms of Fatigue in Professional Dancers
Phase: NA
Conditions: Fatigue, Stress, Job
Sponsor: Poznan University of Physical Education

Study title: Effects of Probiotics on Gut Microbiota, Endocannabinoid and Immune Activation 
and Symptoms of Fatigue in Dancers 
NCT number: NCT05567653 
Document date: 07/28/2025 
 
Study Protocol 
1. Study Population Characteristics 
A total of 51 female dancers initially expressed interest in participating in the study. From this 
group, participants aged 18 to 36 years, engaging in more than 8 hours of dance training per 
week and meeting the inclusion criteria were selected. Twenty-five individuals were excluded 
before randomization due to recent injuries, chronic diseases, the use of probiotics, prebiotics, 
or synbiotics, hospitalization, recent travel to tropical regions, or use of antibiotics, 
cannabinoid-based products, or corticosteroids within the past three months. 
Ultimately, 26 dancers met the inclusion criteria. However, six withdrew early, citing 
scheduling conflicts and an inability to reconcile participation with training and personal 
commitments. Three participants did not attend scheduled blood/fecal collection appointments 
or collect the supplement/placebo. Thus, only 20 attended baseline sampling. Three of these 
later failed to complete the 12-week supplementation protocol. Although they participated in 
baseline sampling, they did not begin supplementation and provided no reason for withdrawal. 
Seventeen dancers completed the full intervention without reporting adverse effects. 
Data from two were excluded during analysis: one due to an outlying BMI (overweight) and 
another for outlier values in biochemical markers (e.g., overdouble AEA levels). Ultimately, 15 
participants (10 placebo, 5 probiotic) were included in the final analysis of serum markers 
(endocannabinoid and inflammatory markers), and 16 in fecal metabolomics (11 placebo, 5 
probiotic). In the latter, only the participant with extreme BMI was excluded. 
Maintaining participation proved difficult due to the demanding schedules of 
professional dancers. Long hours (often 8:00–20:00), performances, and irregular sleeping 
patterns made fasting morning appointments for blood collection challenging. Delivering stool 
samples on time and adhering to stable dietary habits over the 3-month intervention added 
further complexity. Only female students from the university's dance faculty met participation 
requirements. Recruitment attempts from external dance schools or theaters failed due to 
occupational demands or reluctance toward biological sample collection. As only two men 
applied, the final sample was restricted to women to avoid gender-related confounding effects 
in microbiome analysis. 

 
2. Study objectives 
 
The primary objective of this study was to evaluate the effect of a 12-week probiotic 
supplementation with Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 on 
gut microbiota composition, the gut metabolome, and endocannabinoid system markers 
including anandamide (AEA), cannabinoid receptor type 2 (CB2), and fatty acid amide 
hydrolase (FAAH) in professional dancers. Secondary objectives included the assessment of 
changes in serum inflammatory markers such as lipopolysaccharide (LPS), TNF-α, IL-1β, and 
IL-10, as well as the evaluation of gastrointestinal symptoms according to the ROME IV 
criteria, sleep quality measured by the Pittsburgh Sleep Quality Index, fatigue assessed with the 
Fatigue Assessment Scale, pain threshold determined by algometry, and coping strategies with 
stress assessed using the Mini-COPE questionnaire. 
 
3. Study Design 
This exploratory study was conducted as a randomized, double-blind, placebo-controlled trial. 
Participants were randomly assigned to either a probiotic or placebo group using block 
randomization (block size = 4), managed by the supplement manufacturer. Both participants 
and researchers were blinded to group allocation. The 12-week intervention consisted of daily 
supplementation with a probiotic containing Lactobacillus helveticus R0052 (Rosell-52, 
CNCM I-1722) and Bifidobacterium longum R0175 (Rosell-17, CNCM I-3470), commercially 
available as Sanprobi Stress (Sanprobi, Poland). Each capsule contained 3×10^9 CFU and was 
taken in the morning. Placebo capsules matched in shape and mass and contained maltodextrin 
and corn starch. Participants were instructed not to alter their diet or training habits and to report 
deviations such as illness, medication, or dietary changes. Adherence was monitored through 
daily intake diaries and return of unused capsules. All analyzed participants consumed all 84 
doses. 
 
4. Assessment Methods 
 
Anthropometric and Functional Measures 
Baseline assessments (1 week prior) included weight, height, BMI, and body composition (fat 
mass, lean mass, muscle mass) in resting conditions. Blood morphology (WBC, LYM) was 
measured via flow cytometry (Synergy 2 SIAFRT, BioTek) to screen for infections and assess 

immunological and nutritional status. All participants were active dancers or students training 
≥8 hours/week. Physical activity level was self-reported. Handgrip strength was assessed using 
a dynamometer (three trials per hand; average recorded). Pressure pain threshold was measured 
on the thumb flexor muscle using algometry (average of three values). Dietary intake was 
assessed through 3-day food diaries (2 weekdays, 1 weekend day), analyzed via Nuvero 
software to quantify energy, macronutrients, fiber, and cholesterol. Diet quality was assessed 
with the 14-item Mediterranean Diet Assessment Tool (MDAT) and verified through interview. 
No significant baseline dietary differences were found between groups. 
 
Biochemical Analysis of Serum Markers (ECS, LPS, Cytokines) 
Fasting venous blood samples (10 mL) were collected pre- and post-intervention. Serum was 
analyzed using ELISA kits (SunRed Biotechnology, China). CB2 and FAAH levels were 
assessed (sensitivity: CB2 \~0.285 ng/mL; FAAH \~0.116 ng/mL). AEA and LPS levels were 
measured using separate kits (AEA sensitivity \~7.125 ng/mL; LPS \~10.725 EU/L). Cytokines 
measured included TNF-α, IL-1β, and IL-10 (sensitivities \~2.827 ng/mL, \~15.013 pg/mL, 
\~9.012 pg/mL respectively). Intra- and inter-assay CVs were <10% and <12%, respectively. 
 
Fecal Metabolomics 
Stool samples (\~10–30 g) were self-collected and stored at –18°C at home, then transferred to 
–80°C. Untargeted metabolomic analysis was performed using LC–MS (UHPLC Sciex 
ExionLC with TripleTOF 6600+). Separation used a 45-minute gradient on a Phenomenex Luna 
Omega Polar C18 column. Mass spectra (m/z 50–850) were acquired using SWATH. 
Identification used Sciex OS v3.3.1, All-In-One library, NIST, and an internal lab database. 
Mass error tolerance was \~2 ppm. 
 
Psychosocial and Subjective Measures 
Validated questionnaires included: Pittsburgh Sleep Quality Index (PSQI; score 0–21), Fatigue 
Assessment Scale (FAS; 0–32), Mini-COPE (28 items; 14 strategies; grouped into active 
coping, avoidance, emotional support), and a Rome IV-based GI symptom questionnaire. 
 
Statistical Analysis Plan (SAP) 
1. Blood Biomarkers and Questionnaires 
Analyses were performed in STATISTICA 13.3. Normality was assessed using the Shapiro–
Wilk test. Between-group differences: Student’s t-test or Mann–Whitney U. Within-group pre-

post comparisons: paired t-test or Wilcoxon. Two-way repeated measures ANOVA was used to 
assess time, group, and interaction effects (η² effect sizes, Bonferroni post-hoc correction). 
Correlations between AEA, LPS, and other variables: Pearson or Spearman coefficients. p < 
0.05 was considered statistically significant. 
 
2. Metabolomic Data 
Data were filtered to remove low stability signals (QC CV > 25%) and outliers (5th–95th 
percentile), log10-transformed, and analyzed using MetaboAnalystR 4.0. PCA visualized 
global variance; ASCA tested group and time effects (1000 permutations). Mixed models 
(Kenward–Roger correction) evaluated group × time interaction. Univariate t-tests (FDR-
adjusted) identified differentially abundant metabolites. Measurement stability was confirmed 
using quality control (QC) samples. Results were interpreted as exploratory. 
 
Ethics 
The study was approved by the Bioethics Committee of Poznań University of Medical Sciences 
(Approval No. 412/22). Written informed consent was obtained from all participants. The study 
was registered at ClinicalTrials.gov (NCT05567653) and conducted in accordance with the 
Declaration of Helsinki and CONSORT 2010 guidelines. No protocol amendments were made.