Table 1 Descriptions of the study outcome measures

 Time-to-exhaustion during treadmill endurance run

During an endurance treadmill run performed at the heart rate (HR) corresponding to 85% maximal rate of oxygen consumption (V̇O₂max), the participant will run until voluntary time to exhaustion (reflected by meeting 2 out of 3 criteria: respiratory quotient ratio (VCO₂/VO₂) ≥ 1.1; HR reaches a minimum of 85% of age-predicted maximal HR (220 minus age); rating of 17 on the 6 to 20 point Borg Rating of Perceived Exertion scale (BRPE).

 Blood chemistries

Capillary blood samples will be collected before, during, and after the treadmill endurance test to determine concentrations of glucose and lactate. These metabolites will be quantified using commercial, handheld colorimetric devices (ContourNext EZ glucose meter, LactatePlus lactate meter).

 Body composition

This variable will be assessed using the scale (to assess weight) and BOD POD®, a body composition measuring device that measures fat mass and lean muscle mass. Coefficients of variation are 12%, and ICC are 0.9 [20].

 Borg Rating of Perceived Exertion Scale (BRPE):

Exertion is a subjective estimate of exercising intensity. This self-reported instrument assesses effort and fatigue due to physical work [21]. Ratings range from 6=no muscle effort to 20=maximal muscle effort possible, cannot work any harder.

 Bristol Stool Scale (BSFS):

Is a 7-point, self-rated scale of changes in stool form (hardness, shape, water content) that corresponds to intestinal transit time (r=−0.65) [22]. This tool is being used to monitor responses to the intervention and is supported for use in gastrointestinal research.

 Daily Questionnaire:

Is a self-administered, online, daily questionnaire. The items include muscle soreness, consumption of supplements other than the study supplement, and compliance. There are also questions on cold/flu symptoms (used in previous trials) [23].

 Gastrointestinal Severity Rating Scale (GSRS):

This assesses the severity of gastrointestinal symptoms.

The GSRS scores 15 items evaluated in a 7-point Likert scale (1–No discomfort to 7–Very severe discomfort). The questionnaire is composed of a total score (sum of scores from all 15 items) and five subscales: abdominal pain (questions regarding abdominal pain, hunger pains, and nausea), constipation syndrome (questions regarding constipation, hard stools, feeling of incomplete evacuation), diarrhea syndrome (questions regarding diarrhea, loose stools, urgent need for defecation), indigestion syndrome (questions regarding rumbling, bloating, burping, gas) and reflux syndrome (questions regarding heartburn, acid regurgitation). Scores for each question are averaged to provide the sub-scale score. Reliability of the subscales is relatively good, with the Cronbach’s alpha ranging 0.61 to 0.83 [24].

 Digestion-associated Quality of Life Questionnaire (DQLQ):

The impact of GI symptoms on quality of life among healthy adults is measured with this survey [25]. The DQLQ reflects the sum from 9 statements scored on a 10-point Likert scale (0 = never to 1 = always). This questionnaire has high convergent validity with GSRS-QOL scores r=0.54. Test-retest reliability between the DQLQ and GSRS were reported as ICC=0.89 [25].

 Immune and Stress Biomarkers:

Salivary levels of secretory immunoglobulin A (sIgA), alpha-amylase, and cortisol are biomarkers of systemic immune function and stress. Biomarkers will be quantified using ELISA methods using commercial kits.

 Short Healthy Eating Index (sHEI):

This instrument is designed to assess nutrient intake [26].

This is a self-report instrument designed to assess quality of diet relative to the American Dietary Guidelines. This validated instrument assesses 22 items to characterize dietary intake and yields a score between 0 and 100. The higher the score, the more the dietary quality aligns with the American Dietary Guidelines.

 Fiber Screener:

This is a free-access tool created by NutritionQuest [27]. The range of possible results is between 15 g of fiber daily to 54 g of fiber daily. Higher grams of fiber consumed daily is associated with better general health. Validity was assessed using Spearman rank-order correlation coefficients between this short food screener and a full-length Food Frequency Questionnaire. Fruits and vegetable correlations were reported as r =0.71.

 Behavioral Regulation in Exercise Questionnaire (BREQ-3):

This assesses the motivation for exercising. The questionnaire operationalizes motivation into six weighted scales: external regulation, introjected regulation, identified regulation, intrinsic regulation, integrated regulation and amotivation—which count with four items. Positive scores are associated to intrinsic motivation, which is better on the long term for exercising, while negative results are associated to extrinsic and lack of motivation. The questionnaire has demonstrated factorial reliability that was assessed using confirmatory factor analysis with LISREL 8.51 [28].

 Stool Biochemistries

Probiotic strain recovery will be performed on stool samples before and after the intervention to support associations between outcomes and the probiotic. DNA will be extracted from homogenized stool samples using the ZymoBIOMICS 96 MagBead DNA kit (Zymo Research, cat# D4308). The absolute quantification is achieved using the CFX384 Touch Real-Time qPCR Detection System (Bio-Rad Laboratories) according to previously described methods [29] using specific forward and reverse primers.