A Multi-Center, Double-Blind, Randomized, Parallel Group Study to Evaluate the Effect of Fermented Milk Containing Lacticaseibacillus Paracasei Strain Shirota (LCS) on the Upper Airway Symptoms of Triathletes Both Before and After a Competition
Phase: NA
Conditions: Respiratory Tract Infections (RTI), Mucosal Immunity, Systemic Inflammation, Depressive Disorder, Exercise-Induced Immunosuppression, Athletic Performance, Upper Respiratory Tract Symptoms, Mental Health, Cytokine Response, Probiotic Therapy
Sponsor: Federal University of São Paulo

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A MULTI-CENTER, DOUBLE-BLIND, RANDOMIZED, PARALLEL GROUP STUDY 
TO EVALUATE THE EFFECT OF FERMENTED MILK CONTAINING 
LACTICASEIBACILLUS PARACASEI STRAIN SHIROTA (LCS) ON THE UPPER 
AIRWAY SYMPTOMS OF TRIATHLETES BOTH BEFORE AND AFTER A 
COMPETITION  
 
 
 
 
 
Unique Protocol ID- ClinicalTrials.gov  
7.349.261 
 
 
09/05/2025 
 
 
 
 
 
 
 

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FEDERAL UNIVERSITY OF SÃO PAULO (UNIFESP)  
DEPARTMENT OF OTORHINOLARYNGOLOGY-HEAD AND NECK SURGERY  
 
 
 
 
 
 
A MULTI-CENTER, DOUBLE-BLIND, RANDOMIZED, PARALLEL GROUP STUDY 
TO EVALUATE THE EFFECT OF FERMENTED MILK CONTAINING 
LACTICASEIBACILLUS PARACASEI STRAIN SHIROTA (LCS) ON THE UPPER 
AIRWAY SYMPTOMS OF TRIATHLETES BOTH BEFORE AND AFTER A 
COMPETITION  
 
 
 
 
 
 
RESEARCHERS: DR. CESAR MIGUEL MOMESSO DOS SANTOS  
PROF.DR ANDRÉ LUIS LACERDA BACHI  
PROF.DR JÔNATAS BUSSADOR DO AMARAL 
PROF. DR. MAURO VAISBERG  
 
 
 
 
 
 
 
SÃO PAULO - SP  
2025 

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ABSTRACT  
Triathlon, as a sport that combines high-intensity and long duration, imposes the body to an 
expressive physiological adaptation. However, when the challenge exceeds the physiological 
adaptation capacity, it is possible to observe the occurrence of several disorders in the athlete's 
health. In this sense, among several factors that can help in this adaptation, it has been pointed 
out that the microbiota, which can be modulated by diet, age, medical treatments, lifestyle, and 
physical exercise, can favor the maintenance of a healthy status. According to the literature, 
both probiotics, prebiotics, and symbiotics are able to benefit people in different ways, for 
instance, by favoring immunoregulation, these elements can impact the prevention or decrease 
in the frequency and/or intensity of infections in the upper airways, both in the population in 
general and in athletes. However, it is noteworthy to mention that there is still a lack of reports 
in the scientific literature on the effects of the ingestion of fermented milk containing 
Lacticaseibacillus paracasei strain Shirota (LcS), a well-known probiotic agent, not only on 
the modulation of the immune/inflammatory responses, but on the manifestation of symptoms 
of airways and mood state in triathletes. Therefore, in this study, we aimed to investigate, in a 
general way, the effect of daily intake of fermented milk containing 40 billion LcS in amateur 
triathletes, emphasizing that our primary endpoint will be to evaluate the concentration of 
salivary 
IgA, 
and the secondary endpoints 
will be to 
assess 
both 
systemic 
immune/inflammatory and upper airway responses, as well as the incidence and duration of 
upper airway symptoms, and also the mood states. In order to respond to these endpoints, 
samples of saliva, nasal swab, and blood, as well as data regarding quality of life, upper airway 
symptoms manifestation, and mood states will be obtained on five different occasions: 30 days 
(1) and 24 hours before the competition (2), and also immediately (3), 72 hours (4), and 14 
days after the competition end (5). It is important to highlight that the data related to the 
quality of life, upper airway symptoms manifestation, and mood states will be obtained by 
using specific and validated questionnaires, such as SF-36 (Short Form Health Survey 36), 
WURSS-21 (Wisconsin Upper Respiratory Symptom Survey – 21), and BRUMS (The Brunel 
Mood Scale), respectively. Finally, it is also paramount to clarify that the triathletes 
participating in this study will be oriented to ingest a daily bottle of fermented milk containing 
40 billion LcS or placebo/unfermented milk throughout the study period, starting the ingestion 

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30 days before the competition, soon after the first sample collection, until 14 days after the 
competition.  
Keywords: Triathlon; Immunity; Probiotics; Antibodies; Cytokines; Upper Airway´s 
Symptoms; Mood States.  
 
 

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INTRODUCTION  
 Since the last century, studies have shown a close association between the immune system and 
physical exercise (Padilha, 2022). In fact, even though this interaction can promote remarkable 
mutual benefits, some effects can also lead to the loss of a healthy state (Walsh, 2011). For 
instance, it was reported that physical exercise presents a modulating effect on the 
hematological parameters of athletes, particularly a leukocytosis observed after a strenuous 
and prolonged exercise training session, such as a marathon and triathlon (Costa Rosa 
&Vaisberg, 2002; Momesso Santos, 2022). Beyond this observation, it was also shown that 
individuals submitted to sessions of high-intensity and exhausting physical exercises present 
an increase in the incidence of upper respiratory tract infections (URTI), in special when the 
exercise training volume exceeds adequate physiological limits (Costa Rosa &Vaisberg, 2002; 
Pyne, 2000), thus suggesting a possible relationship between training load and the occurrence 
of infections (Gleeson M. , 2000).  
Although it has been pointed out that the URTI incidence rate in amateur athletes 
practitioners of different sports modalities is similar to those observed in the general 
population, when assessed in longitudinal studies both retrospectively and prospectively 
(Keaney, 2021; Orysiak, 2019; Cantó, 2018; Pollastri, 2021), it is noteworthy to mention that 
when the athletes aim to increase their physical performance, in a general way, they have to 
increase the training volume at intensities that generate some level of physiological stress, 
which is often above physiological tolerance. This situation can lead to the development of 
immunosuppression, which increases the possibility of them presenting URTIs in nonseasonal 
periods, mainly in periods close to competitions. In this sense, Walsh et al. (2011) reported that 
swimming athletes tend to present symptoms of URTI in the weeks before competitions, 
whereas in other sports, such as long-distance running, this set of symptoms tends to be more 
frequent after competitions, as observed in our previous study (Vaisberg, 2019).  
According to Bermon (2007), the symptoms of URTI can be multifactorial and the agents' 
promoters of such conditions are not only related to infectious conditions but also 
inflammatory and allergic events. Studies reported that 30% of URTI cases were associated 
with the presence of infectious agents, confirmed by analysis of secretions obtained from the 

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throat and nasopharynx, and the other cases were associated with inhalation of cold and dry air 
(Spence, 2007; Cox, 2008).  
Besides the exposure to these different factors, such as pathogens and cold and dry air, as 
formerly mentioned, the intensity and duration of the physical exercise session, as well as 
climatic conditions, nutritional aspects, mental stress, and other behavioral factors also can 
impact the immune response, thus increasing the possibility to the triggering of URTI. It is 
paramount to highlight that diet is a pivotal element in the modulation of immune and 
inflammatory responses in all individuals, in special those engaged in exercise training 
programs, mainly with exhaustive characteristics, since some agents are able to reduce the 
frequency and magnitude of URTIs (Walsh, 2011).  
 Among some dietary agents studied in this context, probiotics have drawn attention, since it 
was observed that, when the probiotic strains are properly applied, they can positively 
influence the athletes, with remarkable impacts not only in physical capacity but also in the 
performance of athletes (Smarkusz, 2017; Giron, 2022).  
In this respect, the lactobacilli are the probiotic strain most used and it is characterized as 
non-pathogenic, lactic acid bacteria that are resistant to the action of gastric juice and bile 
(Baken, 2006; Shida, 2011; Matsuzaki, 2007). Regarding to its action, a benefit of lactobacilli 
for human health is associated with its corollary ability to regulate the activity of the immune 
system. In a general way, it has been shown that some lactobacilli strains modulate the immune 
response to a Th1 profile with evident production of IL-12p70, which favors the improvement 
of immunity against infections and cancer, whereas other strains mainly induce the production 
of IL-10 driving to a regulatory profile, demonstrating its anti-inflammatory activity (Shida, 
2011). It has been demonstrated that lactobacilli act on cellular responses by increasing not 
only the recruitment and phagocytic capacity of macrophages but also the activation of Natural 
Killer (NK) cells and T cell functions, as well as in hormonal responses through the decrease 
of auto-antibodies (Baken, 2006; Matsuzaki, 2007; Gui, 2020).  
According to Shida et al. (2011), specifically in terms of the supplementation with 
Lacticaseibacillus paracasei strain Shirota (LcS), formerly classified as Lactobacillus casei 
strain Shirota, there are three hypotheses to explain their action on the immune response. In the 

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first hypothesis, the M cells in Peyer's patch, upon uptaking LcS, allowed its recognition 
through Toll-like receptors (TLRs) by macrophages and dendritic cells, inducing their 
activation with the consequent secretion of IL-10. A second mechanism proposes that dendritic 
cells would be able to directly phagocytize bacteria in the gut, thus inducing the secretion of 
IL-10 and TGF-b, leading to the differentiation of T lymphocytes to the most regulatory profile 
of the immune response, and increasing the number of regulatory T lymphocytes (Tregs) in the 
intestinal mucosa. Finally, the M cells presented in the Peyer's patch, by facilitating the 
recognition of LcS by macrophages and dendritic cells, could induce the secretion of IL-
12p70.  
In terms of the effects of lactobacilli supplementation in a physical exercise context, 
although they are still scarce, a longitudinal study carried out with the supplementation of 
Lactobacillus rhamnosus for 3 months found a decrease in duration and severity, but not in the 
incidence of URTI in athletes (Kekkonen, 2007). Beyond this report, Gleeson et al. (2011), 
observed a reduction in the frequency of URTI in association with an increase in salivary 
levels of immunoglobulin A (IgA), in athletes supplemented with LcS for about 4 months. 
Moreover, our group was able to demonstrate that marathon runners supplemented with 
fermented milk containing 40 billion of LcS for a period of 30 days before the marathon, not 
only presented a lower incidence and duration of upper respiratory symptoms (URS) but also 
highlighted the ability of this bacteria in modulating the immune/inflammatory responses, both 
systemic and in the upper airway, specifically after the marathon performance (Vaisberg, 
2019).  
Like the marathon race, the triathlon is recognized as a high-intensity sport, in which the 
athletes are submitted to a strenuous and prolonged competition with remarkable physiological 
impacts (O′Toole M.L., 1989). In this sense, triathletes are particularly prone to muscle injuries 
and fatigue (Kienstra C.M., 2017), not only as a result of mechanical but also inflammatory 
stress, which can induce muscle damage associated with this sport modality (Howatson G., 
2008). Furthermore, triathlon competition also stimulates the production of reactive oxygen 
species (ROS) due to excessive aerobic respiration, ischemia-reperfusion, and leukocyte 
activation (He F., 2016) in response to the pattern of long duration and exposure to 
environmental conditions that favor dehydration, as well as the depletion of carbohydrates can 

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contribute to the occurrence of fatigue, gastrointestinal (GI) problems, hyperthermia, and 
hyponatremia that, in conjunction, can drive to a decrease of athletes’ performance during this 
endurance exercise session (Jeukendrup, 2011). Interestingly, it was reported that central 
fatigue related to the central nervous system (CNS) can be alleviated with the maintenance of 
glycemia through different energy sources (Nybo, 2003). Based on these pieces of information, 
it is possible to consider that it has been well established in the literature that performance in 
the triathlon competition is closely influenced by the state of oxidative stress, inflammation, 
and energy balance.  
Beyond these aspects, it is crucial to cite that in the 80' years of the last century, Morgan 
(1980) popularized the use of a profile mood state (POMS) in the sports context, since he 
identified a close association between successful athletic performance and an "iceberg" mood 
profile, which combined the vigor above-average, with anger, confusion, depression, fatigue, 
and tension below-average. Based on these facts, a new field of research was provided, and the 
subsequent reports reinforced the proposal that mood state is one of the most important 
precompetitive predictive aspects of sports performance and could be a decisive factor in 
athletes’ results (Beedie, PC, & AM., 2000). Recently, Lochbaum and colleagues (2021) 
suggested that analysis of mood state by the parameters of depression, confusion, and vigor 
can be useful to define athletic performance when measured prospectively.   
Particularly in terms of female athletes, the literature highlights the importance of 
monitoring not only psychological aspects but also hormonal variation in order to understand 
their influence on performance, since it has been demonstrated that females in the mid-luteal 
phase showed reductions in both maximal test (Julian, 2017) as well as in prolonged exercise 
performance (Xanne, 2003). Moreover, the phase of the menstrual cycle (follicular and luteal 
phases) and the presence of premenstrual syndrome (PMS) can also impact the inflammatory 
condition related to mood states and stress hormones in female athletes, as previously reported 
by our group (Foster, 2019). Corroborating these pieces of information, Gaion and Vieira 
(2011) reported that the occurrence of PMS leads to negative changes in the mood states of 
athletes during the premenstrual period, which can be correctly assessed through the 
determination of circulating levels of sex hormones (estrogen and progesterone).  

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According to the literature, the mood has been composed of six states: tension, depression, 
anger, confusion, vigor, and fatigue. Moreover, it can be defined as a set of feelings, ephemeral 
in nature, with the possibility of varying in intensity and duration and usually involving more 
than one emotion. In an interesting way, mood presents an evaluative component, particularly 
related to the degree to which the feelings are perceived such as pleasant or unpleasant, in 
association with an arousal component characterized by varying degrees of activity (Lane & 
Terry, 2000).   
Among the (patho)physiological mechanisms related to mood disorders, alterations in both 
hypothalamic-pituitary-adrenal (HPA) axis and in inflammatory/immune responses have been 
highlighted over the last few decades, besides (Caspi, et al., 2003; Mayberg, 1997; Haroon, 
Raison, & Miller, 2012; Bao, 2019). There is a growing of pieces of evidence highlighting the 
role of the association of inflammation in mood modulation, which seems to correlate with all 
of the other proposed mechanisms, such as the neurotrophin, neurotransmitters, and the HPA 
axis (Haroon, Raison, & Miller, 2012; de Miranda, 2020).  
As suggested by Mucci (2020) the increase in the systemic levels of some proinflammatory 
cytokines, Interleukin (IL)-1β, IL-6, interferon-γ (IFN-γ), and tumor necrosis factor-alpha 
(TNF-α), are associated with alterations the mood. In fact, it has been reported that systemic 
inflammation can elicit neuroinflammation through glial activation (Campbell & MacQueen, 
2006) since it is well-known that cytokines can cross the blood-brain barrier and stimulate 
immune and endothelial cells to produce inflammatory mediators (Haroon, Raison, & Miller, 
2012).  
It has been suggested that the intestinal microbiota can affect the levels of stress hormones 
released by the HPA axis, which consequently can also modulate the inflammatory/immune 
response. In this sense, there is a handful of evidence showing the modulatory effect of 
probiotics on neurotransmitter signaling and neuronal activation in the brain, as well as in the 
modulation of hormones and other essential molecules in the body, such as cytokines (Bravo, 
Julio-Pieper, Forsythe, & al., 2012; Appleton, 2018).  
In order to exemplify this prominent modulatory characteristic, particularly in terms of the 
probiotic LcS, it was reported by Otaka and collaborators that the daily administration of 80 

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billion LcS to depressed patients was able to improve the symptoms of depression (Otaka, 
2021). Corroborating this report, Zhang and collaborators showed that the daily administration 
of 10 billion LcS to depressed patients not only demonstrated the ability to improve both 
constipation and depressive symptoms but as well as was able to significantly reduce the 
systemic IL-6 levels (Zhang, 2021).  
In a general way, the scientific literature highlights that alterations in neuroendocrine and 
neurogenesis pathways, synaptic and plasticity functions, and immune/inflammatory responses 
are involved in depression pathogenesis (Zhang, 2021; Wu, 2022; Ribeiro, 2022). Specifically 
in terms of the immune/inflammatory responses, it has been documented that depressive 
patients present high levels of inflammatory cytokines, such as IL-1β, IL-6, and  
TNF-α, and also that this elevation can be an important role in major depression pathogenesis 
(Zhang, 2021; Rudzki, 2019; Yun, 2021).   
As formerly mentioned, alterations in immune/inflammatory responses, which involves a 
remarkable increase in the systemic levels of some cytokines, such as IL-1β, IL-6, and TNF-α, 
also can be observed during exercise training performance, preferentially in exercise sessions 
with high-intensity or strenuous and prolonged, which can lead to an increase in the possibility 
of those athletes to present URTIs (Walsh, 2011; Vaisberg, 2019). Moreover, it was also cited 
that the daily administration of 40 billion LcS was able to mitigate the systemic elevation of 
proinflammatory cytokines in amateur athletes after a performance of a marathon race 
(Vaisberg, 2019).  
Taking these pieces of information together, in which both depressed patients and athletes, 
particularly after exhaustive physical exercise sessions, presented a systemic proinflammatory 
state, it is reasonable to consider that the administration of probiotics, such as LcS, not only 
can affect athletes' mood states, similarly as depressed patients, but also the 
immune/inflammatory responses after a competition of triathlon, which is classified as a 
strenuous and prolonged physical exercise session.  
Triathlon is a sport that combined different types of physical exercises, such as swimming, 
cycling, and running in sequence, which are separated by two transition moments, between 
swimming and cycling, named transition 1, and another between cycling and running, named 

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transition 2. Of interest, this sport can be performed in different distances, ranging between 
short and long distances, which are defined and classified by the International Triathlon Union 
(ITU), the institution that organizes this modality around the world. In order to clarify these 
differences, a short distance is usually composed of 750 meters of swimming, followed by 20 
km of cycling, and, finally, 5 km of running, whereas a standard distance is composed of 1,500 
meters of swimming, followed by 40 km of cycling, and 10 km of running, and a long distance 
is composed of a minimum distance of 1,900 meters of swimming, followed by 90 km of 
cycling, and 21 km of running.  
Based on the previously mentioned, swimming is the first exercise modality in the triathlon 
competition, being performed in rivers, seas, and lakes, which imposes the athletes to different climatic 
conditions, such as water salinity levels, turbulence, and temperature. In this last case, it is noteworthy 
to point out that when the temperature is below 21oC, athletes can wear neoprene clothes up to 5 mm 
thick, but in situations with water temperature below 14oC the distance can be reduced in order to 
reduce exposure to the cold and possible occurrence of hypothermia, or even the swimming stage can 
be canceled (Bentley, McNaughton, Lamyman, & Roberts, 2003). At the end of the swimming 
stage, the athletes proceed to the transition area, where they will have to equip themselves to 
start the cycling stage, this time being counted as race time. 
In the cycling stage, the athlete must pedal on his own without being behind the other 
athlete or a group of athletes, in order to avoid the athlete to face less air resistance, which can 
influence the high economy of movement, generating a lower degree of effort to sustain the 
same performance that, consequently, will lead to the increase of performance in the next 
competition stage. Hausswirth et al. (2001) observed that in this stage, the benefits from the 
vacuum situation are associated with a decrease in oxygen consumption, heart rate, and lactate 
concentration, when compared to the situation without a vacuum. At the end of the cycling 
stage, the athletes return to the transition area, where they will store cycling equipment and 
prepare for the next and last stage, that is, the race.  
Therefore, the race is performed with the accumulation of fatigue generated in the previous 
stages, promoting a high energy cost, which will be superior to the demand necessary to 
sustain the same speed as compared to an isolated race (Hausswirth C. &., 2001). 

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It is utmost of importance to clarify that due to the influence of one stage on the others, 
triathlon competition must be considered a unique modality and not the grouping of three 
different modalities. Thus, the athletes need to perform an exercise training program that 
allows them to perform all these modalities with the optimal physical and physiological 
conditions, which can lead them to achieve the best performance in the competition.  
Based on the literature, although several aspects of triathlon competition already having 
been reported, there are still few studies that aimed to evaluate metabolic changes and 
immune/inflammatory responses in triathletes.  
Among the studies that focused on this issue, Momesso et al. (2022) evaluated the impacts 
of triathlon competition in extreme conditions, and the authors found significant alterations not 
only in the systemic lipid profile and inflammatory status but also in whole blood cell culture 
stimulated or not with lipopolysaccharide (LPS), a pro-inflammatory agent. In view of these 
findings, the adoption of strategies that could mitigate the negative impacts associated with the 
performance of a strenuous and prolonged competition on those athletes, such as dietary 
interventions, for example, using probiotics, can be useful. Corroborating this proposal, Huang 
et al. (2019) analyzed the effect of supplementation with Lactobacillus plantarum PS128 in a 
group of triathletes, and the authors highlighted that this bacteria strain, by its expressive 
ergogenic potential, can be a useful strategy to the athletes achieving better exercise training 
management, physiological adaptations to this physical exercise and health promotion. 
Moreover, the same authors reported that the Lactobacillus plantarum PS128 supplementation 
was associated with an improvement in athletes' performance through microbiota and 
metabolic modulation, but not maximal oxygen consumption (Huang W. C., 2020).  
Taking these pieces of information together, it is clear that the understanding of the 
mechanisms involved in the triggering of disorders in systemic and upper airways, particularly 
in the sport context, is still of great importance, since both in professional and non-professional 
competitive sports, there is a large increase in the number of practitioners. Furthermore, the 
evaluation of the effects of supplementation with probiotics, specifically LcS, on athletes 
submitted to a strenuous and prolonged exercise session, such as a triathlon, not only can 
improve 
our 
knowledge 
concerning 
the 
association 
between 
alterations 
in 

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immune/inflammatory status and susceptibility to the manifestation of upper airway 
symptoms, but, depending on the results found, will allow clarifying whether the 
supplementation with this probiotic can be useful to prevent or even modulate those alterations 
that can lead to systemic and upper airway disorders in triathletes.  
OBJECTIVES  
PRIMARY  
Investigate the effect of daily ingestion of fermented milk containing 40 billion LcS on the 
changes in the concentration of salivary IgA in amateur triathletes participating in this study, 
before and after an official triathlon competition.  
SECONDARY  
Evaluate the effect of daily intake of fermented milk containing 40 billion LcS on both 
systemic immune/inflammatory and upper airway responses, as well as the incidence and 
duration of upper airway symptoms, and also on mood states in amateur triathletes 
participating in this study, before and after an official triathlon competition.  
TRIAL DESIGN  
The study is designed as a multi-center, randomized, controlled, researcher and volunteer 
blinded, with two parallel groups (intervention and placebo). In addition, the randomization 
will be performed as block randomization with a 1:1 allocation. 
MATERIAL AND METHODS  
Registration and adherence to SPIRIT standards  
This study will follow SPIRIT (Standard Protocol Items: Recommendations for 
Interventional Trials) guidelines for randomized clinical trials, and results will be reported 
following 2010 CONSORT (Consolidated Standards of Reporting Trials) standards 
(http://www.consort-statement.org/). In addition, it is important to mention that we intend to 
register the study on ClinicalTrials.gov as soon as this proposal of study is approved and also 
perform the sequential dating of each protocol version in order to mitigate potential confusion 
over which document is the most recent.  

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Population and design of the study 
In order to perform this research project, it is proposed to recruit and select 70-80 amateur 
triathletes, between 25 to 45 years, both sexes, residing in the city of São Paulo, Brazil, who 
will be participating in the same triathlon competition. 
Regarding the sample size, it was determined using G*Power software (version 3.1.9.6). 
The analysis was conducted using the F tests - ANOVA: repeated measures, within-between 
interaction option to accommodate the mixed design of the study, which includes two groups 
and five repeated measurements. It is important to mention that to carry out the sample size 
calculation alignment with the study's primary objective, which is associated with the salivary 
IgA, we used the data presented in the study performed by Vaisberg et al., 2019. In agreement 
with this data, the following parameters were used to calculate the required sample size: Effect 
size (f): 0.15 (Vaisberg et al., 2019). Alpha error probability (α): 0.05. Power (1-β error 
probability): 0.85. Correlation among repeated measures: 0.5. Nonsphericity correction (ε): 1. 
Based on these inputs, the total sample size required to achieve the desired power of 0.85 is 62 
participants. Thus, 70-80 athletes will be included in this study. This sample size will be 
distributed equally between the two groups (35-40 participants per group). 
It is noteworthy to clarify that we intend to separate the volunteers into two groups: male 
amateur triathletes, which will be composed of 40-45 individuals, and female amateur 
triathletes, which will be composed of 30-35 individuals. This discrepant number is associated 
with the fact that the number of male participants in the triathlon competition is higher than the 
number of female participants. For instance, in 2022, the triathlon competition "IronMan 70.3 
São Paulo" presented a total number of 1858 participants, of which 1237 completed the 
competition. In fact, whereas 1027 men completed the competition, only 210 women conclude 
this same competition (available in https://www.ironman.com/im703-sao-pauloresults). Based 
on these pieces of information, we decide to not separate equally the number of volunteers to 
compose the groups of male and female individuals. In order to achieve the number of 
volunteers´ groups (male and female) we will invite the athletes subscribed to the triathlon 
competition through direct contact with the athletes´ consultancies, and also by sending the 
invitation to their social media. 

15 
 
All individuals will be initially evaluated through clinical examinations, and it will exclude 
the volunteers who: (1) report intolerance to lactose; (2) are under any type of pharmacological 
treatment; (3) report chronic rhinopathy; (4) have autoimmune diseases; (5) report the 
occurrence of non-airway recurrent infections. Besides, (6) whether any data from a volunteer 
is missing, this participant will be excluded from the study. As an inclusion criterion, it will 
inquire if the participants already presented the occurrence of any upper airway symptoms 
after an official triathlon competition. In this sense, according to Andersen et al. (2013), the 
incidence of diseases in triathletes is similar to other individual sports, such as marathons and 
swimming, with an incidence of approximately 30% being reported. It is worth clarifying that, 
according to the same authors, any health problems that are not related to the musculoskeletal 
system are considered a disease in this context, particularly in terms of the occurrence of 
similar symptoms of flu post-competition.  
It is also important to cite that the participants will be instructed not to consume products 
containing probiotics or other fermented drinks during the study, and if any volunteer reports 
the regular habit of consuming such products, he will be oriented to interrupt this consumption 
for, at least, two weeks before the start of the study (a washout period). 
In addition, volunteers invited to perform in this study will receive all the information 
related to the present study and those to agree in participate will be oriented to sign the 
Informed Consent Term, which must be previously approved by the Research Ethics 
Committee of the Federal University of São Paulo (UNIFESP), Brazil. The study is in 
agreement with both the Ethical Standards and the Declaration of Helsinki.  
It is important to clarify that there will be no official data monitoring committee established 
for this study. Thus, the only institution responsible for following the development of the study 
is the Ethics Committee, through regular reports. 
In order to improve the volunteers´ adherence to the present study we will offer and provide 
the results concerning the performance of each one in the physical tests, especially concerning 
the cardiopulmonary test, as well as those related to the laboratory tests. All physical and 
laboratory tests are following described. 

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Anamnesis, physical examination, and competition  
At the beginning of the study, all participants will undergo an anamnesis with a 
cardiovascular and otorhinolaryngological analysis to exclude the presence of cardiovascular 
diseases and upper airway symptoms resulting from rhinosinusitis, rhinopathies, or any other 
pre-existing alteration in these systems. In addition, the WURSS (Wisconsin Upper 
Respiratory Symptom Survey) questionnaire will be used to verify the occurrence of upper 
airway symptoms not only during this period but also throughout the study period (Annex 1). 
and the presence of premenstrual syndrome will be self-reported by the female volunteers 
during the anamnesis.  
It is noteworthy to cite that the volunteers will be oriented to maintain their usual daily 
routine, including sleep, diet, and training/physical activity during the study performance. In 
this respect, to characterize not only the quality of life (Annex 2) but also the level of physical 
activity (Annex 3), questionnaires will be applied with open questions about the participants' 
lifestyle and habits, history and current habits of physical activity, as well as participation in 
competitions. Beyond these questionnaires, it will be also assessed the occurrence of 
discomfort or gastrointestinal problems related to the daily intake of fermented milk containing 
LcS or not by using a questionnaire with open questions about gastrointestinal problems will 
be used (Annex 4). The volunteers who reported any gastrointestinal discomfort during the 
ingestion of the fermented milk containing or not LcS will be oriented to immediately interrupt 
this ingestion and seek medical assistance. In case of the volunteer needs to interrupt the 
ingestion of fermented milk for five consecutive days, following medical recommendation, 
this volunteer and its data will be excluded from the study. 
It is utmost of importance to point out that all athletes will have medical follow-ups by Dr. 
Mauro Walter Vaisberg, a specialist in Sports Medicine, throughout the study. Besides, through 
social media, all volunteers will be able to directly contact not only Dr. Mauro Vaisberg but 
also other researchers involved in the study´s development in order to communicate any 
adverse events and other unintended effects of trial interventions.  

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Evaluation of the External Training Load   
The external training load will be determined by the sum of time dedicated to each training 
modality (swimming, cycling, and running), besides the time spent in each of the intensity 
zones (light, moderate and intense) during the training weeks.  
Evaluation of the Internal Load of Training  
The internal training load will be established by the relationship between the time dedicated 
to training and the subjective perception of the effort of the session (PSE-session), which is 
determined through the use of the subjective effort scale modified by Foster et al. (1998), as 
proposed by Impellizzeri et al. (2004).  
Soon after the exercise session, the Borg CR-10 (Borg Category-Ratio) scale adapted by 
Foster (1998) will be presented to athletes (Annex 5).   
Therefore, the internal training load values obtained from this relationship will be expressed 
in arbitrary units (AU).  
Cardiopulmonary Test  
The physical capacity of each volunteer will be characterized through a cardiopulmonary 
test that will be performed on a treadmill attached to the gas analyzer, with 
electrocardiography and gas analysis software. This test will be performed at the Research and 
Training Olympic Center, an institution belonging to the Municipal Secretariat of Sports, 
Leisure, and Recreation of the City of São Paulo. 
In order to carry out the cardiopulmonary test, the patient's preparation rules will be 
followed, which include hair removal from the place where the electrodes will be placed, 
cleaning the skin with alcohol, and scarification with water sandpaper number 400. The 
placement of the electrodes will follow the described by Salient (2019) for the formation of the 
12 leads in the Einthoven triangle.  
The cardiopulmonary test will be performed with a ramp protocol, with a fixed incline of 
1% and load increment (1km/h) every minute until the maximum speed of the treadmill, at 
which time the increment of 2% incline begins every minute. The protocol should be started 

18 
 
with 10.9 or 8 km/h according to the time for a 10 km race, with the time below 35', between 
35' and 46', and above 46', respectively. Tests should be maximum, being interrupted by 
limiting symptoms / intense physical fatigue.  
The data obtained in this test in conjunction with body composition will be used to stratify 
the volunteers into four different strata. In addition, the randomization of volunteers in each 
strata will follow the process: firstly, the researcher JBA, will be responsible for creating a 
computer-generated random list of numbers for the treatment groups of either LcS or placebo, 
and after each volunteer will be anonymously and sequentially allocated in these groups 
according to their recruitment ordering number in the program. 
LcS ingestion  
After stratification, the volunteers will be separated into two groups: (1) LcS Group (LcSG), 
which will ingest 1 bottle of fermented milk (80 grams) with 40 x 109 of LcS per day; and (2) 
placebo/unfermented milk Group (PLG), which will consume 1 bottle of unfermented milk (80 
grams), due to the absence of LcS. Both groups will begin the ingestion 30 days before the 
competition, maintaining it until 14 days after the competition. As previously mentioned, all 
volunteers will be oriented to stop drinking immediately and seek medical attention if any 
symptoms of gastrointestinal disturbance are perceived. To ensure the development of a 
double-blind study, all bottles used in the study will be kindly provided by YAKULT S/A 
Industria e Comércio, Brazil. The bottles will be named A and B by the company and will have 
the same taste, appearance, and smell. Information on the presence or absence of LcS in the 
bottles will be accessed only after all analyzes have been performed. In addition, to monitor 
the adherence of volunteers to the study´s protocol, we intend not only to request the return 
flask fortnightly (empty or not) but also to maintain routine contact via social media with the 
volunteers. This contact also will allow us to provide responses and orientations to any 
questions by them, as well as, weekly, to perform auditing concerning the study´s conduction. 
It is important to clarify that the Yakult S/A Industria e Comércio, Brazil, will provide the 
fermented milk containing LcS and also the placebo/unfermented milk. In addition, Yakult 
Honsha Co., Ltd. Japan, will fund the development of the study, which includes all the 
materials needed to perform the collection, procedures, storage, and laboratory evaluations of 

19 
 
the biological samples obtained on all occasions described in the study. Besides, it is 
noteworthy to mention that the design, management, data check and analysis, and preparation 
of the reports/articles of the study are entirely under the responsibility of the researchers. 
Regarding the ingestion period, it is based on the study by Vaisberg et al. (2019), which 
showed a remarkable capacity of fermented milk containing LcS in modulating several 
immune/inflammatory parameters, both systemic and in the upper airways, in runners after a 
marathon.  
In order to obtain the best quality in our results, we will follow the experimental design 
described below (Figure 1), highlighting that some procedures will be performed on the same 
day of sample collection. 
 
 
 
 
 
 
 
 
 
 
Triathlon competition 
It is important to clarify that the volunteers will perform the same triathlon competition, 
which will be part of the annual calendar for this type of sport. In this respect, it is worth 
mentioning that we intend to carry out the study by inviting the athletes that will perform the 
IronMan 70.3 São Paulo in 2025, which, in a general way, occurs in September, and is 

20 
 
composed of 1,900 meters of swimming, followed by 90 km of cycling, and 21 km of running 
(https://www.ironman.com/im703-sao-paulo). 
Besides, to motivate and ensure that all volunteers will perform the competition with 
maximal effort, the official time of each volunteer to end the competition, obtained through the 
data available by the organizing committee of the competition, will be used to define their 
ranking in the present study and, consequently, the best triathletes in this competition. 
Sample collection 
Blood, saliva, and nasal swab will be collected on five different occasions: (1) before the 
beginning of the ingestion of fermented milk containing LcS or placebo; (2) 24 hours before 
the triathlon competition; (3) immediately (IM); (4) 72 hours (post-72h); and (5) 14 days after 
the competition, as shown in Figure 1. In addition, on the same occasions, the occurrence of 
upper airway symptoms and mood states will also be assessed by using questionnaires. 
Collection of blood samples  
Blood samples will be collected in fasting condition, from an antecubital vein in tubes 
containing or not EDTA anticoagulants. After blood clotting or not, the tube will be 
centrifuged at 2500 rpm for 10min to obtain 500 l of serum or plasma, which will be stored at 
-80o.C for later use to determine circulating cells and serum/plasmatic parameters, as described 
below.  
Blood cell count  
Blood cell count will be carried out on the same day using blood obtained in a tube 
containing EDTA. All subjects must be hydrated at the time of collection. Cell counts 
(erythrocytes, leukocytes, and platelets) will be performed using the automated system Coulter 
brand AcTDiff model (Beckman Coulter, USA).  
Whole blood culture  
A volume of 100L of the blood sample will be mixed with 900L of culture medium, 
containing 10% fetal bovine serum + RPMI 1640, and will be incubated for 48 hours in 48 
well plates, maintained in an air-conditioned oven with 95% oxygen and 5% carbon dioxide at 

21 
 
37o.C. At the same time point, another well containing the same mixture of blood and medium 
will be submitted to stimulation with lipopolysaccharide (LPS) and maintained under the same 
conditions as previously reported. After the incubation period, the culture supernatant will be 
collected and centrifuged at 1400 rpm for 10 minutes, and a volume of, at least, 500L will be 
frozen at 80o.C for further evaluation of cytokine concentration.  
Saliva sample collection  
Two milliliters (2 mL) of saliva samples will be collected from each volunteer directly in 15 
mL sterile Falcon tubes, without stimulation. After collection, each sample will be maintained 
at 4o.C until to be centrifuged at 3000 rpm for 10 minutes at 4o.C. After that, 500L of the 
supernatant will be transferred to 1.5 mL conical tubes and frozen at -80oC for further later 
determination of the concentration of cytokines, IgAs, and antibacterial peptides, without 
adding a preservation medium.  
Assessment of the nasal mucosa infiltrate   
Determination of the percentage of infiltrating cells in the nasal mucosa will be carried out 
with samples obtained from a swab introduced into each nostril of the volunteer and the cells 
obtained will be placed on a slide, followed by their fixation with methanol (99% Synth), and 
then stained in hematoxylin, for later analysis by an experienced pathologist.  
Determination of the concentration of IgAs and antimicrobial peptides  
The salivary concentration of IgAs and the antibacterial peptides [LL-37 (cathelicidin), 
defensin-1, lactoferrin, and lysozyme] will be determined using ELISA commercial kits 
(Biosay Technology Laboratory, Shanghai, China), in accordance with the manufacturer's 
instructions.  
Determination of cytokine concentration  
 The concentration of cytokines IL-1, IL-1ra, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL13, IL-
17A, and TNF-alpha, both in serum, saliva, as in the whole blood culture supernatant, 
stimulated or not with LPS (Lipopolysaccharide), will be determined by using LEGENPlex for 
human cytokines, which is based on a bead system (BioLegend, San Diego, CA, USA), 
following manufacturer's instructions. The cytokine concentrations will be calculated from the 

22 
 
values of their respective standard curves, obtained at the same time as the evaluation of the 
samples.   
Immune cell immunophenotyping  
Evaluation of the number, type, and activation profile of NK cells and T lymphocytes will 
be carried out with blood samples collected in tubes containing EDTA anticoagulant that will 
be mixed 1:1 with Ficoll-Paque (density gradient 1077, FicollPaqueTM PLUS, GE Healthcare, 
Sweden). After centrifugation for 30 minutes at 2000 rpm, the cell pool, corresponding to the 
fraction of mononuclear cells will be collected, counted, and 1 x 105 cells will be added in 1.5 
mL conical tubes. Then, these cells will be washed with MACSBuffer [PBS (1x) containing 
bovine serum albumin (0.5%) and EDTA (2 mM)]. After this step, the cells will be incubated 
with a mix of monoclonal antibodies to evaluate the type, concentration, and activation profile 
of these cells, as shown in Table 1, by using the antibodies to the surface molecules: CD3, 
CD4, CD8, CD16, CD27, CD38, CD45, CD56, CD70, CD197, and HLA-DR.  
 
Table 1. Description of the panel of antibodies to be used in immunophenotyping of NK cells 
and T lymphocytes.  
 
Data acquisition will be performed using a BD Canto cytometer (Becton Dickinson 
Immunodiaginostisc System, San Josem CA, USA). The definition of the gates will be based 
on the analysis of the FMO (Fluorescent Minus One).  

23 
 
Cytotoxicity Assay of NK cells   
The in vitro assessment of the activity of NK cells will be carried out by using the 
commercial kit NKVUE tubes, which allows for evaluating the activity of NK cells through 
the production of interferon-gamma (Nederby, 2018). In addition, the cytotoxic activity of NK 
cells will be also assessed by using another test, which will be initiated by the isolation of 
these cells from peripheral blood using the commercial Invitrogen Dynabeads Untouched 
Human NK Cell kit (Thermo Fisher Scientific, Invitrogen, Vienna, Austria), which allows the 
isolation of these cells by negative selection. Then, using the commercial kit NKTEST, we will 
evaluate the cytotoxic activity of NK cells isolated by mixing these with K562 target cells, in a 
ratio of 5:1 and 2:1, following the manufacturer's guidelines.  
Determination of the systemic concentration of BDNF and hormones   
Serum levels of BDNF (pg/mL), cortisol (ngmL-1), and hGH (mLU mL-1) will be 
determined using ELISA commercial kits [BDNF Quantikine ELISA kit (Catalog number: 
SBD00); Cortisol ELISA EIA-1887 (Catalog number: SKGE008B; hGH ELISA EIA-3552 
(Catalog number: SG100) R&D System, Inc. USA]. In addition, serum levels of sex hormones 
[progesterone (pg/mL), estrogen (pg/mL), and testosterone (pg/mL)] will also be determined 
by ELISA commercial kits [Testosterone Parameter Assay Kit (Catalog number: SKGE010), 
Estradiol Parameter Assay Kit (Catalog number: KGE014), Human Total Progesterone ELISA 
(Catalog number: DYC5415-5), R&D System, Inc. USA]. It is noteworthy to clarify that these 
parameters will be evaluated in all volunteers (both in men and women groups), whilst, in 
particular, the data obtained in the evaluation of the sexual hormones progesterone and 
estrogen also will be used to define the phase of the menstrual cycle in which each female 
volunteer of the study will be during the five different occasions proposed in the present study. 
All evaluations will follow the manufacturers' guidelines.  
Assessments of upper airway symptoms  
The WURSS-21 questionnaire (Wisconsin Upper Respiratory Symptom Survey – 21 items) 
(Annex 1) will be used to assess the occurrence of upper airway symptoms. This instrument is 
composed of 21 questions, in which the first question is associated with the individual's 
perception of "how sick do you feel today?", followed by 10 questions based on specific 

24 
 
symptoms of the cold condition, more 9 questions related to the impact of cold symptoms in 
the individual's quality of life, and one last question comparing the current status of the cold 
symptoms with the previous day. All items will be scored on a Likert scale ranging from 0 to 7 
(from 0 = no symptoms to 7 = severe).  
The severity of the upper airway symptoms will be measured from the sum of the scores of 
all the days in which it was evaluated, with the final score divided by the number of days in 
which there were reports of symptoms. In terms of the severity in relation to the interference in 
the volunteer's quality of life, it will be obtained the sum of the scores of all the days in which 
there was a report, and the final score will be divided by the number of days with symptoms. 
In addition, to analyze the percentage of incidence of the group, the number of days with 
symptoms of each group will be calculated, adjusting for the number of volunteers in the 
group. Then, the adjusted values of each group will be added, considering this value as 100%, 
from which the percentage of each group will be calculated.  
Assessment of mood state  
The Brunel mood scale (BRUMS, Annex 6) will be used to assess the mood state. The 
BRUMS involves 24 mood indicators, divided into six subitems, namely anger, confusion, 
depression, fatigue, tension, and vigor. The volunteer should indicate on a scale from 0 (zero) 
to 4 (four) what they are feeling on the time-point of application of the BRUMS. (Rohlfs, 
2008).  
Statistical analysis  
All data obtained during the development of this study will be added to a unique file, 
preferentially in an Excel file, which will be available to all researchers not only to consult and 
check the data quality/accuracy but also to add any results through GOOGLE-DRIVE. It is 
noteworthy to clarify that, to perform all the statistical analysis proposed here, we will follow 
the "intention-to-treat" (ITT) analysis, not only because it is recommended as an analysis 
strategy for randomized studies but also to be possible to maintain the sample size, as well as 
to use the data obtained from the participants regardless of whether they complete the study 
since ITT considers all randomized participants in the analysis, whether they drop out or not. 
In addition, the multiple imputation (MI) method will be applied to handle missing data since 

25 
 
MI fills in missing values by generating plausible numbers derived from distributions of and 
relationships among observed variables in the data set. Beyond these analyses, in order to 
check and increase the reliability of the results obtained in the ITT analysis, we also intend to 
conduct the statistical analysis regarding the per-protocol (PP) analysis, which will be used 
exclusively the complete data obtained from the volunteers. 
After all data will be added to the Excel file and the data quality/accuracy is checked by the 
researchers, these data will be initially compared with the Gaussian curve, and normality 
determined for each variable.  
If the variable presents a parametric behavior, the mean and standard deviation will be used, 
and they will be compared by using the ANOVA test for repeated measures with post hoc tests 
for intragroup (longitudinal) and intergroup (cross-sectional) analysis.   
On the other hand, if the data present a non-parametric behavior, they will be represented by 
median and interquartile ranges, and they will be compared by using Friedman's test with 
Müller-Dun post hoc test for intragroup (longitudinal) and the Kruskal-Wallis test with Müller 
Dunn's post hoc test for intergroup (cross-sectional).  
Using Pearson (for parametric values) or Spearman tests (for non-parametric values), the 
correlation between the variables studied will be evaluated. 
In addition, the McNemar Chi-square test (with Yates correction) will be used to determine 
whether the difference in the number of volunteers who presented or not upper airway 
symptoms, as well as in the duration of the symptoms, is significantly different in each group 
(paired analysis). Additionally, Chi-square test will be used to perform the same analysis but 
between the volunteer groups (unpaired analysis).  
A risk of less than to 0.05 (p<0.05) will be considered statistically significant for every 
study. 
 
Researchers´ information and contributions  
First of all, it is important to mention that the researchers' names and affiliations are: 

26 
 
- 
Jônatas Bussador do Amaral (JBA), from the Federal University of São Paulo (UNIFESP) 
and the sponsor-investigator (Address: Rua Pedro de Toledo, 781, 1o. andar, sala 13, Vila 
Clementino, 04039-032, São Paulo, Brazil, e-mail: amaraljb@gmail.com). 
- 
César Miguel Momesso dos Santos (CMMS), from United Metropolitan Colleges, Centro 
Universitário FMU. 
- 
André Luis Lacerda Bachi (ALLB), from Santo Amaro University (UNISA)  
- 
Mauro Vaisberg (MV), from the Federal University of São Paulo (UNIFESP)  
In addition, the researcher's contributions are: CMMS, ALLB, and JBA wrote the first draft 
of the study. CMMS, ALLB, JBA, and MV will conduct the study. CMMS and MV will be 
responsible for the recruitment and selection of volunteers, for the application of anamnesis, 
physical examinations, evaluation of the external and internal training load, and 
cardiopulmonary test. MV will be responsible for the clinical follow-up of the volunteers. JBA 
will be responsible for the volunteers' randomization. CMMS, ALLB, and JBA will be 
responsible for the collection, procedure, and storage of the biological samples, as well as for 
the performance of laboratory tests. CMMS and ALLB will be responsible for conducting the 
primary statistical analysis. All researchers equally will contribute to the refinement of the 
study protocol and will participate in the preparation of the report(s) and manuscript(s).  
WORK PLAN AND SCHEDULE  
For the performance of the present study, we propose a work plan and schedule that will 
need 12 months to be developed. Thus, a scale of monitoring and collection of materials will 
be recommended, which initially will depend on the availability of each volunteer recruited to 
carry out the long-term triathlon competition.  
Thus, we propose the following work plan and schedule:  
- Recruitment of athletes and performance of clinical evaluations on all volunteers included 
in the study;  
- Collection of blood, saliva, and nasal swab samples before starting the daily intake of 
fermented milk containing or not the bacteria (LcS group, n=35-40; Placebo group, n=35-
40, respectively).  

27 
 
- study volunteers will ingest the fermented milk for 44 days;  
- Collection of blood, saliva, and nasal swab samples 24 hours before the competition;  
- realization of the competition by all volunteers included in the study;  
- Collection of blood, saliva, and nasal swab samples immediately after the end of the 
competition;  
- Collection of blood, saliva, and nasal swab samples 72 hours after the competition;  
- Collection of blood, saliva, and nasal swab samples 14 days after the competition;  
- Evaluation of the occurrence of upper airway symptoms throughout the study´s period;  
- Performing the laboratory evaluations described in the Material and Methods section; - 
Statistical analysis of the results and preparation of the report. 
SCHEDULE OF ACTIVITIES TO BE DEVELOPED  
Parameters 
Enrollment 
Allocation 
Intervention 
Pre- 
Triathlon 
Post- 
Triathlon 
Timepoint 
-60d to -30d 
-30d 
-1d 
0d 
3d 
14d 
Recruitment 
and 
selection 
of 
volunteers 
X 
 
 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
 
Anamnesis, 
anthropometry, 
cardiopulmonary test 
X 
 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
Intervention: LcS/Placebo intake 
 
 
X 
X 
X 
X 
X 
Questionnaire - WURSS 
X 
 
 
X 
 
X 
X 

28 
 
Questionnaire - IPAQ  
X 
 
 
X 
 
 
 
Questionnaire 
- 
gastrointestinal 
problems 
X 
 
 
X 
 
X 
X 
Questionnaire – SF36 
X 
 
 
X 
 
X 
X 
Questionnaire – BRUMS 
X 
 
 
X 
 
X 
X 
Questionnaire 
- 
Subjective 
Perception of Effort Scale 
X 
 
 
 
X 
 
 
Blood, saliva, nasal swab collection 
 
 
X 
X 
X 
X 
X 
 
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Rohlfs, I. C. (2008). A Escala de Humor de Brunel (Brums): instrumento para detecçãoprecoce da 
síndrome do excesso de treinamento. Revista Brasileira de Medicina do Esporte, pp. 176-181.  

32 
 
Rudzki, L. O. (2019, Oct 16). Probiotic Lactobacillus Plantarum 299v decreases kynurenine 
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Shida, K. N. (2011). Flexible cytokine production by macrophages and T cells in response to probiotic 
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Smarkusz, J. O.-S. (2017). Probiotic strains as the element of nutritional profile in physical activity – 
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Sugama K., S. K. (2015). Changes of thioredoxin, oxidative stress markers, inflammation and 
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Vaisberg, M. P.-C.-D. (2019). Daily Intake of Fermented Milk Containing Lactobacillus casei Shirota 
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Walsh, N. P. (2011). Position statement. Part two: Maintaining immune health. Exercise immunology 
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Wu, S. X. (2022, Oct 28). Possible Effects and Mechanisms of Dietary Natural Products and Nutrients 
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Yun, S. W. (2021, Sep 13). Lacticaseibacillusparacasei NK112 mitigates Escherichia coli-induced 
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Beneficial microbes, pp. 541-551.  
Zhang, X. C. (2021, Jun 29). Effects of Fermented Milk Containing Lacticaseibacillusparacasei Strain 
Shirota on Constipation in Patients with Depression: A Randomized, Double-Blind, PlaceboControlled 
Trial. Nutrients, p. 2238.  
 
 
 

33 
 
ANNEX I – Questionnaire Wisconsin Upper Respiratory Symptom Survey - 21  
 
 
 
 
 
 
 
 

34 
 
 
 ANNEX II – Questionnaire SF36 

35 
 
 

36 
 
 

37 
 
 
 

38 
 
ANNEX III - Questionnaire of Physical Activity (IPAQ)  
848 
NOME...................................................................................................................................  
849 
Data…………………..  
850 
As perguntas a seguir referem-se à quantidade de atividade física que você realizou na semana 
851 
passada.  
852 
Para os 7 dias anteriores, por favor, considere a quantidade, o tipo e a intensidade da atividade física 
853 
que realizaram. Se isso é mais ou menos do que o normal, isso deve ser refletido em suas respostas.  
854 
Este 
tipo 
de 
atividadepodeassumirqualquer 
forma 
e 
podemincluiratividadesdiáriasnormais, 
855 
comocaminharaté as lojas.  
856 
Atividadefísicavigorosarefere-se a atividades que necessitaram de grandeesforçofísico para ser 
857 
realizado/completado, além de exigir que vocêrespirassemais que o normal.  
858 
Atividadesmoderadasreferem-se a atividades que necessitaram de moderadoesforçofísico para ser 
859 
realizado/completado, além de exigir que vocêrespirasse um poucomais do que o normal.  
860 
Com relaçãoaostreinos, tenteclassificarcadasessãointeiracomovigorosaoumoderada.  
861 
Marque de forma adequadanascaixasfornecidas.  
862 
Seja o maishonestopossível.  
863 
1. Nos últimos 7 dias, quantosdiasvocêparticipou de atividadesfísicasvigorosas, comolevantamento de 
864 
peso, atividadesaeróbias, treinamentointenso, ciclismo, nataçãoou corrida?  
865 
 
_________ Dias   
- Se nenhum, responda 00 e passe para a pergunta 3  
866 
2. Nos diasem que você fez a atividadefísicavigorosaquanto tempo vocêcostumagastarfazendoisso?  
867 
 
__________Horas_______ Minutos 
868 
3. Nos diasem que você fez atividadefísicamoderadaquanto tempo vocêcostumagastarfazendoisso?  
869 
 
_________ Dias   
- Se nenhum, responda 00 e passe para a pergunta 4  
870 
__________Horas_______ Minutos 
871 
4. Nos últimos 7 dias, quantosdiasvocêcaminhouporpelomenos 10 minutos de cadavez?  
872 
 
_________ Dias   
- Se nenhum, responda 00 e passe para a pergunta 6  
873 
5. Nos diasem que vocêcaminhouporpelomenos 10 minutos de cadavez, no total, quanto tempo 
874 
vocêgastaandando?  
875 
__________Horas_______ Minutos   
876 

39 
 
6. Quanto tempo você gasta sentado em um dia normal? Pode incluir o tempo de estudo, visitando846 
877 
amigos, lendo ou assistindo televisão.  
878 
__________Horas_______ Minutos 
879 
ANNEX IV - Questionnaire gastrointestinal problems  
880 
Nome.............................................................................................................Data…………......................
881 
. 
882 
Durante a semana passada você se sentiu incomodado por qualquer um dos seguintes sintomas abaixo 
883 
listado? Por favor, indique a sua resposta através da marcação ou preenchimento do quadrante para 
884 
cada um dos seguintes:  
885 
 
886 
1. Algum destes sintomas, já afetou sua capacidade de treinar esta semana?  
887 
Sim ( ) Não ( )  
888 
Se sim, indicar qual das seguintes situações:  
889 
Pequena redução do treino, conseguindo manter o mesmo nível ( )  
890 
Treinamento reduzido abaixo do nível normal ( )  
891 
Parou completamente de treinar ( )  
892 
2. Você já tomou qualquer medicação (mesmos em receita) nesta semana para aliviar 
893 
doenças respiratórias ou sintomas de desconforto gastrointestinal?  
894 

40 
 
Sim ( )  
895 
Se sim, qual o nome da medicação/// 
896 
............................................................................................................................................  
897 
Não ( )  
898 
3. Você se consulta com seu médico sobre seus sintomas da doença esta semana?  
899 
Sim ( )  
900 
Se sim, você tem tomado qualquer medicação prescrita esta semana?  
901 
Nome da medicação 
902 
............................................................................................................................................  
903 
Não ( )  
904 
4. Você fez alguma alteração na sua dieta normal esta semana?  
905 
Sim ( )  
906 
Se sim, o que mudou 
907 
............................................................................................................................................  
908 
Não ( )  
909 

41 
 
ANNEX V – Subjective Perception of Effort Scale 
910 
 
911 
Carga Interna = Valor apontado de PSE x minutos da sessão de treino 
912 
 
913 
 
914 
 
915 
 

42 
 
888  
ANNEX VI – Brunel Mood Scale (BRUMS)  
889  
 

 
 
 
 
 
INFORMED CONSENT FORM 
You are being invited to take part in a study as a volunteer. If you agree to take part in the study, 
please sign at the end of this document, which is in two copies. One copy is yours and the other 
belongs to the researcher responsible. 
RESEARCH INFORMATION: THE EFFECT OF FERMENTED MILK CONTAINING 
LACTICASEIBACILLUS PARACASEI STRAIN SHIROTA (LCS) ON THE UPPER AIRWAY 
SYMPTOMS OF TRIATHLETES BEFORE AND AFTER A COMPETITION 
RESPONSIBLE RESEARCHER: Prof Dr Jônatas Bussador do Amaral. 
CONTACT PHONE/EMAIL: (11) 99418-9999 amaraljb@gmail.com 
 
DESCRIPTION OF THE RESEARCH: 
Because triathlon is a sport that combines high intensity and long duration, it imposes 
significant physiological adaptation on the body. However, when the challenge exceeds the 
capacity for physiological adaptation, it is possible to observe the occurrence of various 
disorders in the athlete's health, such as the manifestation of symptoms in the upper airways. In 
this sense, among the many factors that can help with this physiological adaptation is the 
microbiota, which can be modulated by age, medical treatments, , physical exercise and, in 
particular, by diet, favoring the maintenance of a healthy state. According to the literature, 
probiotics, prebiotics and symbiotics can benefit people in different ways. In fact, due to their 
obvious ability to help regulate the immune/inflammatory system, these agents can have an 
impact on reducing or even preventing the frequency and/or intensity of upper airway 
symptoms, both in the general population and in athletes. However, it is worth noting that there 
is still a lack of reports in the scientific literature on the effects of drinking fermented milk 
containing Lacticaseibacillus paracasei strain Shirota (LcS), a known probiotic agent, not only 
on the modulation of immune/inflammatory responses, but also on the manifestation of upper 
airway symptoms and mood in triathletes. 
Therefore, in this study, we aim to investigate, in general, the effect of daily intake of fermented 
milk containing 40 billion LcS in amateur triathletes, emphasizing that our primary endpoint 
will be to evaluate the occurrence of upper airway symptoms and the secondary endpoints will 
be to evaluate both systemic and upper airway immune/inflammatory responses, as well as 
mood states. To meet these objectives, saliva, nasal swab and blood samples will be obtained, as 
well as data on quality of life, the manifestation of symptoms in the upper airways and mood 
states on five different occasions: 30 days (1) and 24 hours before the competition (2), as well as 
immediately (3), 72 hours (4) and 14 days after the end of the competition (5). It is important to 
note that the data relating to quality of life, the manifestation of symptoms in the upper airways 
and mood states will be obtained using specific and validated questionnaires, such as the SF-36, 
WURSS-21 and BRUMS, respectively. Finally, it is also important to clarify that the triathletes 
taking part in this study will be instructed to drink a bottle of fermented milk containing 40 
billion LcS or placebo (unfermented milk) every day for the duration of the study, 

starting the intake 30 days before the competition, right after the first sample collection, until 
the 13th day after the competition. 
The risks related to the volunteers' participation in this research are minimal. There may be 
some discomfort not only when blood samples are taken, with the possible formation of a small 
hematoma (purple spot), but also when nasal cells are collected using the nasal swab. To reduce 
these risks, samples should always be taken by trained professionals. 
If you agree to participate, you will contribute to an increased understanding of the effects of 
daily ingestion of fermented milk containing 40 billion LcS on the immune/inflammatory 
response and mood state of triathletes before and after a session of prolonged and exhausting 
physical exercise, such as a long-duration triathlon. In addition, it may allow the implementation 
of new health promotion strategies for triathletes by considering fermented milk containing LcS 
as an agent capable of positively and beneficially modulating the immunity, inflammatory 
response and mood of triathletes. 
If, after consenting to participate, you withdraw your consent, you are guaranteed the full right 
and freedom to withdraw your consent at any stage of the research, before or after data 
collection, regardless of the reason and without any prejudice to you. You will not receive any 
remuneration. The results of the research will be analyzed and published, and your identity will 
be preserved and kept confidential. For any further information, you can contact the researcher 
responsible either in person at the Research Laboratory of the Department of 
Otorhinolaryngology and Head and Neck Surgery the Federal University of São Paulo 
(UNIFESP), located at Rua Pedro de Toledo 781, 1º. andar, sala 3, Vila Clementino, CEP: 
04039-032, or by telephone (11) 99418-. 
9999. 
 
São Paulo,  
of  
2025. 
 
 
 
 
 
Participant's signature 
Jônatas Bussador do Amaral 
Responsible for the research 

Page 01 of 18 
 
 
 
 
 
 
 
 
 
 
 
SUBSTANTIATED OPINION OF THE CEP 
 
Prepared by the Co-participating Institution 
 
RESEARCH PROJECT DATA 
 
Research 
Title: 
THE 
EFFECT 
OF 
FERMENTED 
MILK 
CONTAINING 
LACTICASEIBACILLUS 
PARACASEI CEPA SHIROTA (LCS) ON UPPER AIRWAY 
SYMPTOMS IN 
TRIATHLETES BEFORE AND AFTER A COMPETITION 
Researcher: Andre Luis Lacerda Bachi 
Thematic area: 
Version: 3 
CAAE: 78576224.9.3001.5505 
Proposing Institution: FEDERAL UNIVERSITY OF SAO PAULO 
Main Sponsor: Self-financing 
 
OPINION DATA 
 
Opinion number: 7.349.261 
 
Project presentation: 
CEP/UNIFESP project n: 0434/2024 (co-participating center) 
Project initiated by the researchers themselves Local 
manager: Jônatas Bussador do Amaral 
Research team: Prof. Dr. Mauro Vaisberg, Dr. Jônatas Bussador Do Amaral and Cesar Miguel Momesso Dos 
Santos 
Project linked to the Department of Otorhinolaryngology, São Paulo Campus, UNIFESP. 
 
Multicenter study in Brazil. Two research centers will participate, including UNIFESP. 
Coordinating Center: Santo Amaro University (Unisa). Researcher responsible: Andre Luis Lacerda Bachi 
Coparticipating Centers: 02 
 
-The information listed in the fields "Presentation of the Project", "Objective of the Research" and "Evaluation of 
Risks 
and 
Benefits" 
was 
taken 
from 
the 
f 
i 
le 
Basic 
Information 
of 
the 
Research 
(<PB_INFORMAÇÕES_BÁSICAS_DO_PROJETO_ 2321205.pdf> posted on 21/05/2024) and from the file of the 
detailed project sent (<Projeto_Versao_Portugues_Final.pdf> posted on 24/03/2024). 
 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
 Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
0U0F1: SP 
Municipalit  SAO PAULO 
Phone: 
y: 
E-mailcep@unifesp.br 
(11)3385-4343 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page 02 of 18 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
0U0F1: SP 
Phone: 
Municipalit  SAO PAULO 
y: 
E-mailcep@unifesp.br 
(11)3385-4343 
Continuation of Opinion: 7.349.261 
 
 
 
 
 
PRESENTATION: 
Because triathlon is a sport that combines high intensity and long duration, it imposes significant physiological 
adaptation on the body. However, when the challenge exceeds the capacity for physiological adaptation, it is 
possible to observe the occurrence of various disorders in the athlete's health. In this sense, among the various 
factors that can help with this adaptation, it has been pointed out that the microbiota, which can be modulated by 
diet, age, medical treatments, lifestyle and physical exercise, can favor the maintenance of a healthy state. 
According to the literature, probiotics, prebiotics and synbiotics can benefit people in different ways. For example, 
by promoting immunoregulation, these elements can have an impact on preventing or reducing the frequency 
and/or intensity of upper airway symptoms, both in the general population and in athletes. However, it is worth 
noting that there are still no reports in the scientific literature on the effects of drinking fermented milk containing 
Lacticaseibacillus paracasei strain Shirota (LcS), a known probiotic agent, not only on the modulation of 
immune/inflammatory responses, but also on the manifestation of airway symptoms and mood in triathletes. 
Therefore, in this study, our aim will be to investigate, in general, the effect of daily intake of fermented milk 
containing 40 billion LcS in amateur triathletes, emphasizing that our primary endpoint will be to assess the 
occurrence of upper airway symptoms and the secondary endpoints will be to assess both systemic and upper 
airway immune/inflammatory responses, as well as mood states. To answer these endpoints, saliva, nasal swab 
and blood samples, as well as data on quality of life, upper airway symptom manifestation and mood states will be 
obtained on five different occasions: 30 days (1) and 24 hours before the competition (2), as well as immediately 
(3), 72 hours after the competition (4) and 24 hours after the competition (5). 
(4) and 14 days after the end of the competition (5). It is important to note that the data related to 
quality of life, the manifestation of upper airway symptoms and mood states will be obtained using specific and 
validated questionnaires, such as the SF-36, the WURSS-21 and the BRUMS, respectively. Finally, it is also 
essential to clarify that the triathletes taking part in this study will be instructed to drink a bottle of fermented milk 
containing 40 billion LcS or placebo/unfermented milk every day for the duration of the study, starting 30 days 
before the competition, right after the first sample collection, until the 13th day after the competition. 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page 03 of 18 
 
 
(11)3385-4343 
E-mailcep@unifesp.br 
Municipalit  SAO PAULO 
y: 
0U0F1: SP 
Phone: 
ZIP CODE: 04.021- 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
Continuation of Opinion: 7.349.261 
 
 
 
 
 
HYPOTHESIS: 
Supplementation with Lacticaseibacillus paracasei strain Shirota interferes with immune system modulation and 
performance in triathlon athletes. 
 
Research Objective: 
PRIMARY OBJECTIVES 
To investigate the effect of daily intake of fermented milk containing 40 billion LcS on the occurrence of upper 
airway symptoms in amateur triathletes participating in this study, before and after an official triathlon competition. 
 
SECONDARY OBJECTIVES 
To evaluate the effect of the daily intake of fermented milk containing 40 billion LcS on the systemic and upper 
airway immune/inflammatory responses and also on the mood states of the amateur triathletes who took part in 
this study, before and after an official triathlon competition. 
 
 
 
Assessment of Risks and Benefits: 
Regarding the risks and benefits, the researcher declares: 
RISKS: 
The risks for the research volunteers will be minimal, both for the blood collection, because although it is a routine 
clinical procedure that is generally safe, there may be minor discomfort (pain) or hematoma at the puncture site, 
fainting or dizziness, and for the collection nasal cells through the nasal swab. In order to reduce these risks, the 
collection should always be carried out by trained professionals. 
 
BENEFITS: 
Among the benefits for the volunteers is that they can undergo clinical examinations, with the results being given to 
them free of charge, including relevant information for improving the structure of their physical training. 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page 04 of 18 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
0U0F1: SP 
Phone: 
Municipalit  SAO PAULO 
y: 
(11)3385-4343 
E-mailcep@unifesp.br 
Continuation of Opinion: 7.349.261 
 
 
Comments and Considerations on the Research: 
STUDY TYPE: double-blind, randomized, controlled trial with two parallel groups 
LOCATION: Otorhinolaryngology Research Laboratory (ORL-LAB) . Rua Pedro de Toledo, 781, 1st floor room 13 
PARTICIPANTS: 
- Inclusion criteria: 
 
PROCEDURES: 
Proposed Methodology: 
It will be designed as a double-blind, randomized, controlled trial with two parallel groups:LcS Group (LcSG): 
Consumes milk fermented with LcS daily for 30 days before and 14 days after a triathlon competition.Placebo 
Group (PLG): Consumes non-fermented milk daily for the same period.Main characteristics of the 
study:Participants: 60-70 male and female amateur triathletes aged between 25 and 45, living in São Paulo, Brazil. 
Interventions: Daily consumption of fermented milk containing SCM or placebo for 56 days. Measures: Upper 
airway symptoms assessed by the WURSS-21 questionnaire before, during and after competition. Blood, saliva 
and nasal swab samples taken at five different times for various analyses. Cell count Cell culture with LPS 
stimulation to assess cytokine production (IL-1¿, IL-1ra, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL13, IL-17A, and TNF- 
alpha) using the LEGENPlex for human cytokines kit (BioLegend, San Diego, CA, USA). Saliva sample 
Concentration of IgAs [LL-37 (cathelicidin), defensin-1, lactoferrin, and lysozyme] determined by ELISA 
commercial kits (Biosay Technology Laboratory, Shanghai, China).Nasal infiltrate Fixation with methanol 
(99% Synth) and then stained in hematoxylin, for later analysis by an experienced pathologist.Immunophenotyping 
of immune cells Evaluation of the number, type and activation profile of Natural Killer cells and T lymphocytes 
Using antibodies to the surface molecules: CD3, CD4, CD8, CD16, CD27, CD38, CD45, CD56, CD70, CD197 and 
HLA-DR. 
Cytotoxicity of Natural Killer Cells The in vitro evaluation of the activity of Natural Killer (NK) cells will be 
carried out using the commercial NKVUE Tubes kit, which makes it possible to assess the activity of NK cells 
through the production of interferon-gamma. Determination of the systemic concentration of BDNF and 
hormonesCommercial ELISA kits [BDNF Quantikine ELISA kit (Catalog number: SBD00); 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page 05 of 18 
 
 
(11)3385-4343 
E-mailcep@unifesp.br 
Municipalit  SAO PAULO 
y: 
0U0F1: SP 
Phone: 
ZIP CODE: 04.021- 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
Continuation of Opinion: 7.349.261 
 
 
Cortisol ELISA EIA-1887 (Catalog number: SKGE008B; hGH ELISA EIA-3552 (Catalog number: SG100) R&D 
System, Inc. USA]Serum levels of sex hormones [progesterone (pg/mL), estrogen (pg/mL) and testosterone 
(pg/mL) will also be determined by commercial ELISA kits [Testosterone Parameter Assay Kit (Catalog Number: 
SKGE010), Estradiol Parameter Assay Kit (Catalog Number: KGE014), Human Total Progesterone ELISA (Catalog 
Number: DYC5415-5), R&D System, Inc. The physical capacity of each volunteer will be characterized by means of 
a cardiopulmonary test which will be carried out on a treadmill coupled to a gas analyzer with electrocardiography 
and gas analysis software. The following variables will be determined: The participant's physical capacity. 
Measurement of maximum oxygen consumption (VO2max). Anaerobic threshold. Pulmonary ventilation. Heart rate. 
Mood assessed using the Brunel Mood Scale. Occurrence of gastrointestinal discomfort monitored. 
 
INCLUSION CRITERIA: 
Inclusion criteria for the study: 
1. Participation in the IronMan 70.3 São Paulo competition: The main requirement for taking part in the study is to 
be registered and participating in the IronMan 70.3 São Paulo competition in 2024. 
2. Age group: Participants must be between 25 and 45 years old. 
3. Gender: Both men and women can take part in the study. 
4. Resident in São Paulo: Participants must reside in the city of São Paulo, Brazil. 
5. No pre-existing conditions: Participants must not have: Intolerance to fermented milk. Use of any type of 
medication. Chronic rhinitis. Autoimmune diseases. Recurrent infections. 
6. Agreement to follow the study protocol: Participants must agree to: Consume the fermented milk with LcS or the 
placebo daily during the study period. Follow the study instructions. Complete questionnaires and provide blood, 
saliva and nasal swab samples. 
7. Sign the Informed Consent Form: All participants must read, understand and sign the Informed Consent Form 
before starting the study. Observations: The study aims to recruit 60-70 participants, divided into two groups: LcS 
and placebo. Participants will be selected on the basis of inclusion and exclusion criteria. The research team 
reserves the right to exclude any participant who does not comply with the study protocol. 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page 06 of 18 
 
 
Continuation of Opinion: 7.349.261 
 
 
 
 
 
EXCLUSION CRITERIA: 
(1) reported intolerance to fermented milk; 
(2) are under any kind of pharmacological treatment; 
(3) report chronic rhinopathy; 
(4) have autoimmune diseases; 
(5) report the occurrence of recurrent non-airborne infections. In addition, 
(6) feeding during the study of foods containing 
Lactobacilli 
(7) if any data is missing from a volunteer, that participant will be excluded from the study 
(for more information, see detailed design). 
Consideration of mandatory terms of presentation: 
1- The main documents were presented: cover sheet; complete project; copy of the CEP/UNIFESP registration, 
financial budget and timetable. 
2- Other important documents attached to the Brazil Platform: 
HSP-HU/UNIFESP CoEPE letter no. 117/24 
Forwarding letter (, posted on 21/05/2024 ) Letter of consent (, 
posted on 24/03/2024 ) 
Commitment and confidentiality agreement 
Concession agreement 
3¿ The model of the ICF was presented by the researcher. 
4- The questionnaires / interview scripts are attached at the end of the detailed project. 
5 - Project_Portuguese_Version_FINAL_2.docx 
6 - Template_TCLE_Unifesp_Triathletes_2.docx 
7 - Termo_anuencia_instituicao_URC_2024_Andre_Bachi.pdf 
8 - Physiology_Laboratory_Agreement_Term.pdf 
9 - Answers_questions_PB_2_completo.doc 
Recommendations: 
RECOMMENDATION 1- It is the researcher's obligation to develop the research project in full compliance with the 
proposal submitted to the CEP. Changes and alterations to the protocol must be communicated in the form of an 
AMENDMENT through Plataforma Brasil. 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
0U0F1: SP 
Municipalit  SAO PAULO 
Phone: 
y: 
E-mailcep@unifesp.br 
(11)3385-4343 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page 07 of 18 
 
 
Continuation of Opinion: 7.349.261 
 
 
 
 
 
RECOMMENDATION 2- From the date of approval, it will be necessary to send partial reports (every six months) 
and the final report (at the end of the study), by means of a NOTIFICATION on the Brazil Platform. Researchers 
must inform and justify to the CEP any need for temporary suspension or permanent suspension of the research. 
Incidents and adverse events must also be reported to CEP-Unifesp by means of a NOTIFICATION on the Brazil 
Platform. 
 
RECOMMENDATION 3- Researchers should take all necessary precautions when collecting and storing data in 
order to guarantee the secrecy and confidentiality of the research participants' information. It is recommended to 
download the data collected to a local electronic device, deleting any data from virtual platforms or the cloud. The 
research protocol documents (files of forms, terms, data and samples) must be kept for at least 5 years after the 
end of the research. 
 
 
 
Conclusions or Outstanding Issues and List of Inadequacies: 
Response to opinion no. 7.205.814 of November 5, 2024. PROJECT APPROVED. 
 
RESPONSE TO PENDING ISSUES 
 
 
 
PENDENCY 1 - In relation to the timetable provided in the basic information form: we ask that the researchers 
check whether there is a need to change the start date for the "Recruitment and selection of volunteers" and 
subsequent stages, so that there is enough time between the responses and approval by the CEP/UNIFESP. We 
remind you that no study can begin before approval by CEP/UNIFESP (CNS Operational Standard 001 of 2013, 
item 3.3.f). 
ANSWER 1 The dates have been changed in the schedule presented in the Detailed Project and in the Brazil 
Platform. 
PENDENCY ATTENDED TO 
 
 
 
PENDENCY 2 ¿ It is requested that the name of Cesar Miguel Momesso Dos Santos (cited in field 
 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
0U0F1: SP 
Municipalit  SAO PAULO 
ZIP CODE: 04.021- 
Phone: 
y: 
E-mailcep@unifesp.br 
(11)3385-4343 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page 08 of 18 
 
 
Continuation of Opinion: 7.349.261 
 
 
"Assistant", in the basic information form of Plataforma Brasil), be included on the front sheet (cover) of the detailed 
project. 
ANSWER 2: The researcher's name has been added 
PENDING CARRIED OUT 
 
 
 
PENDENCY 3- Inform in the detailed project methodology and in the ICF the average time needed for participants 
to answer all the questionnaires. ANSWER 3: As requested, the average time needed to answer each 
questionnaire in this study has been added to both the main text of the study and the ICF. In this sense, it is worth 
mentioning that it will be necessary: - 5 to 7 minutes to answer the WURSS questionnaire, to check the occurrence 
of upper airway symptoms not only in this period, but also throughout the study period (Annex 1); - 10 to 12 
minutes to answer the quality of life (Annex 2); - 5 to 8 minutes to answer the physical activity level questionnaire 
(Annex 3); - 5 to 7 minutes to answer the questionnaire with open questions about gastrointestinal problems. 
(Annex 04). 
PENDENCY PARTIALLY FULFILLED CEP/UNIFESP ANALYSIS: The average time needed to answer the 
questionnaires was not added to the ICF. 
 
Answer 3: The following text was added to the ICF: ¿It is worth mentioning that it will take 5 to 7 minutes to answer 
the WURSS questionnaire (to check the occurrence of upper airway symptoms), 10 to 12 minutes to answer the 
quality of life questionnaire, 5 to 8 minutes to answer the physical activity level questionnaire and, finally, 5 to 7 
minutes to answer the questionnaire with open questions about gastrointestinal problems¿. 
PENDENCY ATTENDED TO 
 
 
 
PENDENCY 4- - According to the letter of referral, research participants linked to Hospital São Paulo and 
UNIFESP will not be involved. We therefore ask for clarification regarding the sending of the COEPE letter and the 
completion of the CEP registration form stating that UNIFESP outpatient clinics will be used. ANSWER 4: Vale 
 
 
 
 
 
 
 
 
 
 
 
 
 
(11)3385-4343 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
0U0F1: SP 
Municipalit  SAO PAULO 
Phone: 
y: 
E-mailcep@unifesp.br 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page 09 of 18 
 
 
Continuation of Opinion: 7.349.261 
 
 
to clarify that for the development of this study proposal, clinical evaluation will be recommended with emphasis on 
otorhinolaryngological aspects. Therefore, in order to guarantee reliable data for each volunteer taking part in the 
study, we will use the outpatient facilities of the Department of Otorhinolaryngology and Head and Neck 
Surgery at UNIFESP exclusively to carry out this assessment. Finally, it is worth stating that no other UNIFESP 
sector/ambulatory will be used. 
PENDENCY ATTENDED TO 
 
 
 
PENDENCY 5- According to the letter of referral, in addition to the competition venues, participants will also be 
recruited from the Olympic Center and Unisa. Answer 5: With regard to this item, it is worth first clarifying that, as 
presented in the text of the research project, "In order to reach the number of volunteer groups (male and female), 
we will invite the athletes registered for the triathlon competition through direct contact with the athletes' advisors 
and also by sending the invitation to their social media." Therefore, recruitment will be carried out exclusively and 
directly by those responsible for developing the study and there will be no recruitment from the Olympic Center and 
Unisa. However, it is worth noting that the premises of the Olympic Center will be used to carry out the 
cardiorespiratory assessment, while the premises of UNISA will be used to collect, process and store the samples 
necessary for the development of this study, in addition to carrying certain laboratory assessments. 5a) It will be 
necessary to send a letter of consent/authorization from the person in charge of the sites regarding the research 
(one for each site). Answer 5a) As described above, since there will be no recruitment at the Olympic Center and 
Unisa, it is not necessary to send a letter of consent/authorization from the person in charge of the sites regarding 
the research. PENDENCY NOT FULFILLED CEP/UNIFESP ANALYSIS: Since the premises of the Olympic Center 
and UNISA will be used, it is necessary to send the letters of consent and authorization from those responsible for 
the sites. 
Answer 5.a): Letters of consent have been attached to the Brazil Platform system 
PENDENCY ATTENDED TO 
 
PENDING 5.b) If recruitment is carried out through the media, social networks, posters, etc., the text of this 
recruitment must be sent to the CEP for analysis. This information 
 
 
 
 
 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
0U0F1: SP 
Municipalit  SAO PAULO 
ZIP CODE: 04.021- 
Phone: 
y: 
E-mailcep@unifesp.br 
(11)3385-4343 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page1018 
 
 
Continuation of Opinion: 7.349.261 
 
 
should be included in the project methodology. GUIDELINE: Invitations to take part in the research should not be 
made using lists that allow the invitees to be identified or their contact details (e-mail, telephone, etc.) to be viewed 
by third parties. Any individual invitation sent by e-mail can only have one sender and one recipient, or be sent in 
the form of a hidden list. (C I R C U L A R OFFICE 
NO. 
2 / 2 
0 2 1 / C O N E P / S E C N S / M S , 
d i s p o n i v e l 
e m : 
http://conselho.saude.gov.br/images/Oficio_Circular_2_24fev2021.pdf). Answer 5b) The text of the invitation letter to 
be sent to the athletes is shown in ANNEX 06. In addition, as requested, this information has been added to the 
main text of the research project, as shown below. 
In order to reach the number of volunteer groups (male and female), we will invite the athletes registered for the 
triathlon competition through direct contact with the athletes' advisors and also by sending the invitation by e-mail 
or even to their social media. The text to be sent is shown in ANNEX 05 - PENDENCY PARTIALLY FULFILLED 
ANALYSIS CEP/UNIFESP: Correct the number of the annex referring to the letter of invitation (annex VII and not 
VI). Answer 5b) We have changed the numbering of the annex. 
PENDENCY ATTENDED TO 
 
 
 
PENDENCY 6 - We request that the methodology of the detailed project include the justification for the use of 
placebo, as CNS resolution 466/2012, item III.3.b., states that research must "have full justification, where 
appropriate, for the use of placebo, in terms of non-maleficence and methodological necessity, and the benefits, 
risks, difficulties and effectiveness of a new therapeutic method must be tested, comparing it with the best current 
prophylactic, diagnostic and therapeutic methods. This does not exclude the use of placebo or no treatment at all in 
studies in which there are no proven methods of prophylaxis, diagnosis or treatment¿. 
ANSWER 6: As requested, the use of placebo in this study is initially justified by the participant's acknowledged 
belief that the simple use of the bottle containing fermented milk will prevent the manifestation, as well as reduce 
the duration and airway symptoms associated with the triathlon competition. In addition, the placebo will be useful 
for understanding and isolating the biological effects, particularly those related immune/inflammatory 
aspects, considering the non-maleficence, beneficence and justice of 
 
 
 
 
 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
0U0F1: SP 
Municipalit  SAO PAULO 
Phone: 
y: 
E-mailcep@unifesp.br 
(11)3385-4343 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page1118 
 
 
Continuation of Opinion: 7.349.261 
 
 
possible improvements in the manifestation of airway symptoms associated with triathlon competition. Finally, it is 
worth noting that the fermented milk containing SCL is an exclusive product of the company Yakult Honsha 
Co.,Ltd. and only it can supply the placebo for the development of this research project. The above text has been 
added to the main text of the research project, specifically in the sub-item "Ingestion of SCL" under "Materials and 
Methods". 
PENDENCY ATTENDED TO 
 
 
 
PENDENCY 7- Page 18 of the detailed project reads: ¿To ensure the development of a double-blind study, all vials 
used in the study will be kindly provided by YAKULT S/A Indústria e Comércio, Brazil.¿ We request that a letter 
from the company agreeing to provide the vials for research be attached to the Brazil platform. 
ANSWER 7: In response to the request, the letter from YAKULT S/A Indústria e Comércio, Brazil stating that it will 
provide the vials has been attached to the Brazil Platform. 
PENDENCY ATTENDED TO 
 
 
 
PENDENCY 8- We would like to remind you that, according to Resolution 466/2012:III.3.d, if the treatment proves 
to be effective at the end of the study, the same treatment should be made available to the control group: please 
clarify and it will be necessary to include this information in the detailed project and in the ICF. (Resolution 
466/2012:III.3.d, proposes.d, proposes: "Ensure that all participants at the end of the study, by the sponsor, have 
free and indefinite access to the best prophylactic, diagnostic and therapeutic methods that have been shown to be 
effective.").ICF: inform that if the treatment proves to be effective at the end of the study, the same treatment 
should be made available to the control group Answer 8: Regarding this item, it is worth clarifying that: 1) the 
research project will be carried out using a product (fermented milk containing LcS) which is not registered as a 
medicine but as a foodstuff, according to its registration with the Brazilian Ministry of Agriculture, Livestock and 
Supply, attached to the Brazil Platform; 2) the purpose of the study is not to evaluate the possible use of fermented 
milk containing LcS as a treatment for a specific disease or pathological process, but rather to evaluate the 
possible probiotic effect (immunomodulation) of this fermented milk in a specific context, which involves triathlon 
competition; 3) 
 
 
 
 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
0U0F1: SP 
Municipalit  SAO PAULO 
Phone: 
y: 
E-mailcep@unifesp.br 
(11)3385-4343 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page1218 
 
 
Continuation of Opinion: 7.349.261 
 
 
corroborating the item above, as presented in the research project, the study will be carried out with the 
participation of healthy individuals who do not suffer from a particular disease or pathological process. 4) in 
addition, the fermented milk to be used in the study is produced and sold commercially in our territory by the 
company YAKULT S/A Indústria e Comércio, Brazil, and is therefore widely available to the general population; 
Given this information, which highlights specific points involved in the study, if the use of fermented milk containing 
SCL proves to be effective at the end of the study, it can be used by any member of the control group by 
purchasing it commercially, without there being any obligation to make it available to the control group. 
PENDENCY ATTENDED TO. 
 
 
 
PENDENCY 9- In relation to the collection of blood, saliva and nasal swab, in addition to the information provided, 
the following information must be included in the project methodology (according to CNS Resolution 441/12 of May 
2011): 
9.a) and in what quantity (blood); 
Answer 9a: As requested, the following text has been added to the project: "Blood will be collected in 4 tubes 
containing EDTA anticoagulants and in 1 tube without anticoagulants. It is worth noting that each tube can 
accommodate a volume of 4mL of blood, so we will not have a total volume of 20mL of blood from each volunteer 
on each occasion." 
PENDENCY ATTENDED TO 
 
9.b) which professional will carry out the collection and in which location; Answer 9b: As requested, the following 
text has been added to the project: "It is important to clarify not only that the collection of biological material will be 
carried out on the premises of the Santo Amaro University (UNISA) laboratory, under the responsibility of a nursing 
professional, trained and experienced to carry out this task (...)" PENDENCY ATTENDED TO 
 
PENDENCY 9.c) under what conditions and in what form the biological material will be transported to the analysis 
laboratory; Answer 9c: As requested, the following text has been added to the draft: "(...) but also that all material 
collected will be kept refrigerated in a box. 
 
 
 
 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
0U0F1: SP 
Municipalit  SAO PAULO 
Phone: 
y: 
E-mailcep@unifesp.br 
(11)3385-4343 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page1318 
 
 
Continuation of Opinion: 7.349.261 
 
 
Styrofoam with the addition of regular ice or icex until it is processed." PENDENCY PARTIALLY MET 
CEP/UNIFESP ANALYSIS: It is not clear how the samples will be transported from the collection site to the 
analysis laboratory. Transportation must follow RDC 504/2021 
Answer 9.c). The following was added to the project ¿The transportation of biological samples in this research 
(saliva, nasal swab and blood) will take place in strict compliance with the Resolution of the Collegiate Board 
(RDC) No. 504, of May 27, 2021, of the National Health Surveillance Agency (ANVISA). Provision is made for 
collection and packaging procedures, where samples will be collected by a responsible professional and in a 
suitable environment and with all the necessary care to guarantee the integrity and safety of the biological material 
and volunteers. The samples will be packaged in watertight primary containers, individually identified and labeled in 
accordance with technical standards. They will then be placed in absorbent secondary containers, also identified 
and securely closed. The outer packaging will be resistant to impacts and leaks, and will be duly identified with the 
name of the research, the name of the technical manager, the recipient's address and contact information. 
Transportation will be by land, using the researcher's own vehicle. Suitable conditions of temperature, humidity and 
pressure will be guaranteed throughout transportation, according to the nature of the samples. A transportation 
manifest will be issued containing all relevant information about the samples, including the biological risk 
classification. It should be emphasized that all professionals involved in the transportation process will be wearing 
appropriate PPE and have expertise in good practices for transporting biological materials. In the event of a 
biological sample leak during transportation, a contingency plan will be activated immediately. This plan includes 
notifying the relevant authorities, isolating the contaminated area and using appropriate personal protective 
equipment. Containment of the leak will be carried out with specific absorbent materials, followed by rigorous 
cleaning and decontamination. All actions will be recorded and a full incident report will be drawn up. 
PENDENCY ATTENDED TO 
 
PENDENCY 9d- under what conditions the biological material will be stored, in what location, for how long before 
analysis; 
Answer 9d: It is worth noting that we have revised the description of the items mentioned and the following text 
 
 
 
 
 
 
 
 
 
 
 
 
(11)3385-4343 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
0U0F1: SP 
Municipalit  SAO PAULO 
Phone: 
y: 
E-mailcep@unifesp.br 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page1418 
 
 
Continuation of Opinion: 7.349.261 
 
 
was added to the project: "After processing, the samples will be kept, without the addition of preservatives, in a - 
80o.C freezer on the premises of the UNISA Research Laboratory (URC) until they are used in the laboratory 
procedures recommended in the study. The samples will be stored for a maximum of 6 months before being used 
in (...), and will then be disposed of as soon as the study is completed." 
PENDENCY ATTENDED TO 
 
PENDENCY 9.e) where (laboratory) the analysis will be carried out and what will be analyzed; 
Answer 9e: All laboratory evaluations will be carried out at the UNISA Research Center and/or at the Research 
Laboratory of the Department of Otorhinolaryngology and Head and Neck Surgery of UNIFESP. 
PENDENCY PARTIALLY MET 
ANALYSIS CEP/UNIFESP: The response did not mention what will be analyzed. 
Answer 9.e). The following has been added to the project: "In these sectors, the saliva, nasal swab and blood 
samples collected from the volunteers will be processed. 
PENDENCY ATTENDED TO 
 
PENDING 9.f)- what will be done with the material after the analysis (will it be discarded, stored until the end of the 
research, or stored even after the research is finished?). If it will be stored after the end of the research, state 
where it will be stored and for what reason (repetition of the analyses to verify the results, and/or for possible new 
analyses). 
Answer 9f: All samples will be kept in a -80o C freezer until the end of the research project, after which they will be 
disposed of in accordance with biosafety regulations. After the end of the experiments and publication of the 
results, all the samples will be discarded. The biological material will be packed in white bags and collected by the 
São Paulo City Hall and incinerated (following UNIFESP's solid waste disposal rules). 
PENDENCY ATTENDED TO 
 
 
 
PENDENCY 10- - According to Resolution 441/11 Art °1 item II - ¿Biorepository: collection of human biological 
material, collected and stored during the execution of a specific research project, in accordance with technical, 
ethical and operational regulations or standards prior to the implementation of the project. 
 
 
 
 
 
 
 
 
 
(11)3385-4343 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
0U0F1: SP 
Municipalit  SAO PAULO 
Phone: 
y: 
E-mailcep@unifesp.br 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page1518 
 
 
Continuation of Opinion: 7.349.261 
 
 
defined, under the institutional responsibility and management of the researcher, without commercial purposes¿, It 
is requested to include information about the biorepository in the detailed project methodology: 
10.a) Researcher Responsible and email Answer) Researcher Jônatas Bussador do Amaral (email: 
jbamaral@unifesp.br) 
10.b) Department/Sector Answer) Department of Otorhinolaryngology and Head and Neck Surgery 
-EPM/Unifesp. 
10.c) 
Head of Department and email Reply) Prof. Dr. Onivaldo Cervantes (email: 
ocervantes@uol.com.br) 
10.d) Address where the Biorrepository will be located Answer) Located in Unifesp's Research Building 1 (Rua 
Pedro de Toledo 781)-São Paulo-SP 
10.e) Telephone number of the laboratory where the biorepository 
will be located Answer) The ORL Lab's telephone number is 11 
5576-4848 (VOIP 1372). 
10.f) Type of biological material stored Answer) Whole blood and its derivatives (serum and plasma), saliva and 
nasal swab 
10.g) How the samples will be stored Answer) After collection, the biological material will undergo processing 
consisting of: 1-Centrifugation at 800xG 2-Removal of the supernatant 3-Storage in 2mL conical tubes 4-Labeling 
and identification of tubes and boxes 5-Storage in an ultrafreezer - 80º.C In this biorepository, whole blood and its 
derivatives (serum and plasma), saliva and human tissues are stored. All the samples in the biorepository are 
discarded after the end of the projects. They are removed and then disposed of via the infectious solid waste route 
(¿white waste¿) and sent for incineration via the São Paulo city hall. 
10.h) How the samples will be collected Answer) Blood, saliva and nasal swab samples will be obtained by health 
professionals. The blood will be obtained via venipuncture. The saliva will be collected in appropriate tubes. Finally, 
the nasal swabs will be inserted into the nasal cavity and used to make the smear on glass slides, which will then 
be stored in the ORL Lab. 
COPY OF THE AMENDED TEXT OF THE ENTIRE ITEM 10 E(TEXT INSERTED IN THE DETAILED DESIGN AND 
TCLE): 
Biorepository 
The ORL Lab's ultrafreezer keeps biological samples at a temperature of -80oC and is used as a Biorepository. All 
sample preparation and control of the flow of 
 
 
 
 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
0U0F1: SP 
Municipalit  SAO PAULO 
Phone: 
y: 
E-mailcep@unifesp.br 
(11)3385-4343 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
Page1618 
 
 
Continuation of Opinion: 7.349.261 
 
 
storage is done at the ORL Lab under the responsibility of researcher Jônatas Bussador do Amaral (email: 
jbamaral@unifesp.br). Located in Unifesp's Research Building 1 (Rua Pedro de Toledo 781), this biorepository 
belongs to the Department of Otorhinolaryngology and Head and Neck Surgery and is headed by Prof. Dr. 
Onivaldo Cervantes (email: ocervantes@uol.com.br). The ORL Lab's telephone number is 11 5576-4848 
(VOIP1372). This biorepository stores whole blood and its derivatives (serum and plasma), saliva and human 
tissues. All samples from the biorepository are discarded after the end of the projects. In this study, saliva, blood 
and its derivatives (serum and plasma) will be stored. PENDENCY ATTENDED TO 
 
 
 
PENDING 11- TFIC ATTENTION: The TFIC attached is inadequate. Please prepare the TCLE again, using the 
TCLE template on the CEP/UNIFESP website, link: UNIFESP - Pesquisa - Comitê de Ética em Pesquisa ¿ Projeto 
envolvendo seres humanos -Plataforma Brasil: Modelo de TCLE, or at: https://cep.unifesp.br/linksuteis#modelos) 
11.a) In the attached TCLE, in addition to the CEP/UNIFESP data, the data (telephone, e-mail and address) of the 
CEP of the coordinating center should be inserted with the indication that it can be contacted if there is any doubt 
about the ethics of the study; ANSWER 11: In response to the request, the TCLE has been revised and prepared 
according to the suggested model. 
PENDENCY PARTIALLY MET 
CEP/UNIFESP ANALYSIS: The CEP data for the coordinating center has not been added. We request that this be 
done. 
Answer 11): The following was added to the ICF: ¿If necessary, you can also contact the CEP of Universidade 
Santo Amaro (UNISA), at the address: Rua Prof. Enéas de Siqueira Neto, 340 ¿ Jardim das Imbuias ¿ Prédio F1 ¿ 
Térreo ¿ Pós-Graduação do Campus Interlagos ¿ CEP: 04829-300 ¿ São Paulo ¿ SP, telephone (011) 2141-8687 
and email: pesquisaunisa@unisa.br. The UNISA CEP's opening hours are from Monday to Friday, from 8am to 
12pm, and from 1pm to 6pm, except on Friday until 5pm. You can also contact the National Research Ethics 
Council (CONEP) by e-mail:conep@saude.gov.br or by telephone: (61) 3315-5878 to resolve doubts and ask 
for clarification. PENDENCY ATTENDED TO 
 
 
 
 
 
 
 
 
 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
0U0F1: SP 
Municipalit  SAO PAULO 
ZIP CODE: 04.021- 
Phone: 
y: 
E-mailcep@unifesp.br 
(11)3385-4343 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
0U0F1: SP 
Phone: 
Municipalit  SAO PAULO 
y: 
(11)3385-4343 
E-mailcep@unifesp.br 
Page1718 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
Continuation of Opinion: 7.349.261 
 
 
NEW PENDENCY 12- ICF:  Add the data of the researcher Jônatas Bussador do Amaral (UNIFESP) next to the data 
of André Luiz de Lacerda Bachi 
 
The following excerpts have been added to the ICF: 
Investigators: Dr. André Luis Lacerda Bachi (UNISA) and Dr. Jônatas Bussador do Amaral (UNIFESP)¿. 
If you have any questions about the research, you can contact the researchers responsible: André Luiz de 
Lacerda Bachi and Jônatas Bussador do Amaral , on the following telephone numbers: 11 5576-4848 / Voip 1372, 
cell phone (11)99891-7767, Rua Pedro de Toledo, 781, 1º andar sala 13, São Paulo ¿ SP and e-mail 
miguelmomesso@gmail.com¿ 
PENDENCY ATTENDED TO 
 
 
Final considerations at the discretion of the CEP: 
In view of the above, the Research Ethics Committee - CEP, in accordance with the attributions defined in CNS 
Resolution No. 466, of 2012 and/or CNS Resolution No. 510, of 2016, and in CNS Operational Standard No. 001, 
of 2013, expresses its approval of the research protocol. 
 
 
 
 
This opinion has been drawn up on the basis of the documents listed below: 
 
Document Type 
Archive 
Post 
Author 
Situation 
Basic Information 
PB_BASIC_INFORMATION_OF_P 
11/12/2024 
Accepted 
Project 
ROJETO_2321205.pdf 
09:33:01 
Others 
Answers_questions_PB_2_co 
11/12/2024 
Jônatas Bussador do 
Accepted 
mpleto.doc 
09:31:48 
Amaral 
Declaration of 
Physiology_Laboratory_Agreement_Term 
22/11/2024 
Jônatas Bussador do 
Accepted 
Institution and 
Infrastructure 
ogia_.pdf 
09:50:39 
Amaral 
Declaration of 
Institution_annulment_term_URC_2024 
22/11/2024 
Jônatas Bussador do 
Accepted 
Institution and 
Infrastructure 
_Andre_Bachi.pdf 
09:49:43 
Amaral 
TCLE / Terms of 
Template_TCLE_Unifesp_Triathletes_2.doc 
22/11/2024 
Jônatas Bussador do 
Accepted 
Assent / 
x 
09:45:39 
Amaral 
Justification for 
Absence 
Detailed Design / 
Project_Portuguese_Version_FINAL_2.do 
22/11/2024 
Jônatas Bussador do 
Accepted 
Brochure 
Researcher 
cx 
09:45:22 
Amaral 

FEDERAL UNIVERSITY SÃO 
PAULO - UNIFESP 
0U0F1: SP 
Phone: 
Municipalit  SAO PAULO 
y: 
(11)3385-4343 
E-mailcep@unifesp.br 
Page1818 
 
 
Address: 
Rua Sena Madureira, 1500 - 2° andar 
Neighborhood: VILA CLEMENTINO 
ZIP CODE: 04.021- 
Continuation of Opinion: 7.349.261 
 
Others 
Ministry_of_Agriculture_Registry.pdf 
12/10/2024 
Jônatas Bussador do 
Accepted 
16:46:37 
Amaral 
Declaration of 
Collaboration_letter_YAKULT.pdf 
12/10/2024 
Jônatas Bussador do 
Accepted 
Sponsor 
16:40:06 
Amaral 
Others 
CADASTRO_CEP_UNIFESP_assinado. 
21/05/2024 
Jônatas Bussador do 
Accepted 
pdf 
12:04:00 
Amaral 
Others 
Signed_forwarding_letter.p 
21/05/2024 
Jônatas Bussador do 
Accepted 
df 
12:01:19 
Amaral 
Others 
COEPE.pdf 
21/05/2024 
Jônatas Bussador do 
Accepted 
11:58:45 
Amaral 
Others 
Confidentiality_Covenant_ 
24/03/2024 
Andre Luis Lacerda 
Accepted 
Signed.pdf 
09:16:37 
Bachi 
Others 
Signed_Concession_Term.pdf 
24/03/2024 
Andre Luis Lacerda 
Accepted 
09:15:43 
Bachi 
Others 
Signed_Agreement_Letter.pdf 
24/03/2024 
Andre Luis Lacerda 
Accepted 
09:15:06 
Bachi 
TCLE / Terms of 
ECLE_Project_Triathlon_LcS.pdf 
24/03/2024 
Andre Luis Lacerda 
Accepted 
Assent / 
09:14:01 
Bachi 
Justification for 
Absence 
Detailed Design / 
Project_Portuguese_Version_Final.pdf 
24/03/2024 
Andre Luis Lacerda 
Accepted 
Brochure 
Researcher 
09:13:51 
Bachi 
 
Status of opinion: 
Approved 
Needs CONEP appraisal: 
No 
 
 
 
 
 
 
SAO PAULO, January 29, 2025 
 
Signed by: 
Paula Midori Castelo Ferrua (Coordinator)