A total of 24 runners (20 male, 4 female) participated in the study. All participants were required to have run a marathon race quicker than 5 h within the previous 2 years. Participant characteristics are presented in Table 1. All participants were free of medications, such as NSAIDs, antidepressants, or diuretics, nutritional supplements and any history of GI-related medical issues (IBS or abdominal surgery). After explaining the nature and risks of the experimental procedures to the participants, their informed written consent was obtained. The study was approved by the Ethics Committee of Liverpool John Moores University.
Table 1 Participant characteristics Baseline testing
At least 4 weeks before the marathon, and prior to the supplement period, participants visited the laboratory and completed the Gastrointestinal Symptom Rating Scale (Svedlund et al. 1988) to assess baseline GI symptoms. Participants then completed an incremental running test to determine lactate threshold (LT) and peak oxygen uptake (\(\dot{V}\)O2peak) as previously described (Jones 2006). Briefly, participants ran a minimum of six stages on a motorized treadmill (HP Cosmos Saturn, Traunstein, Germany). Each stage was 3 min in duration and interspersed with 30 s breaks to allow blood sampling. Running speed was increased by 1 km h−1 at the end of each stage, until runners reached volitional exhaustion.
Supplement period
After baseline testing, in a double-blind, randomised and matched-pairs design, participants underwent a 28 day period of supplementation consuming either a commercially available probiotic (PRO) or a visually identical placebo (PLC). Participants also consumed an additional supplement capsule on the morning of the race, 2 h before the start. Participants were matched according to their most recent marathon performance (PRO: 222 ± 46 min; PLC 220 ± 40 min) and body mass (Table 1). The probiotic supplement capsules contained the active strains Lactobacillus acidophilus CUL60, L. acidophilus CUL21, Bifidobacterium bifidum CUL20, and Bifidobacterium animalis subsp. Lactis CUL34 (Proven Probiotics Ltd, Port Talbot, Wales). The minimum concentration was 25 billion colony-forming units (CFU) per capsule. The placebo capsules were visually identical and consisted of cornstarch only (Proven Probiotics Ltd, Port Talbot, Wales). Participants were instructed to swallow the capsule daily after their first meal. The randomization code was held by a third party, unlocked for analyses upon sample analysis completion. During the supplementation period, participants were instructed to refrain from all probiotic foods (i.e., fermented yogurts). For the full 28 day supplement period, participants were required to complete a daily training and GI symptom diary. Six GI symptoms were included: bloating, nausea, urge to vomit (upper GI), flatulence, urge to defecate, and stomach cramps (lower GI). Symptoms were scored from 0 to 10 on a visual analogue scale. Symptoms scored ≥ 4 were classified as ‘moderate’ or worse and these data were summed during the supplementary period.
Marathon race
During the 24 h before the race, participants consumed a standardized high CHO, low fibre diet [per kg body mass: 8.0 g CHO (0.28 g fibre); 2.0 g protein; 1.0 g fat]. Compliance to the diet was confirmed with food diaries and the remote food photography method (Martin et al. 2009). After an overnight fast, participants reported to the laboratory at ~ 07:00 h and resting blood samples were taken. Participants were then provided a standardized breakfast [572 kcal; 128 g CHO (4.4 g fibre), 7 g protein, 3.5 g fat, and a minimum of 500 mL water] before a pre-race venous blood sample was collected. Participants performed self-selected warm-ups before a race briefing to reiterate in-race nutrition and subjective measures. The race started at 12:00 pm. Runners ran the 42,195 m race on a synthetic 400 m outdoor track (105.48 laps) which was in close proximity to the laboratory. Weather conditions throughout the race were: temperature: 16–17 °C; wind speed: 8–16 km h−1; precipitation: 0 mm. During the race, heart rate was monitored throughout (Firstbeat Sports©, Jyväskylä, Finland) and subjective ratings of perceived exertion (RPE) (Borg 1970) were recorded every 15 min. Each 400 m lap time was recorded using electronic chips and a timing mat (BibTag System, MYLAPS, USA). Global GI discomfort was assessed every 15 min using a modified Likert scale (Nieman et al. 2006). These physiological and symptomology data were consolidated and reported as the mean of each third of the race distance completed, given that reductions in average running pace and reporting of ‘hitting the wall’ are seen after 25–30 km (Santos-Lozano et al. 2014; Buman et al. 2009), and glycogen depletion is theorised to occur between 32 and 40 km (Locksley 1980).
In-race nutrition
Participants consumed one 60 mL CHO gel (SIS Isotonic Gel, Blackburn, UK) and 200 mL of water 10–15 min before the start of the race and one 60 mL CHO gel with 200 mL of water 40 min after the start of the race and subsequently every 20 min for the remainder of the race. Gels consisted of 22 g maltodextrin and 0.01 g sodium. This provided an average of 66 g h−1 CHO in 180 mL and 600 mL h−1 of water during the race, a strategy that has been shown to improve performance in non-elite runners relative to a self-selected strategy (Hansen et al. 2014). To familiarize with this nutritional strategy, participants were informed of the strategy and provided with identical gels to practice this during their two longest training runs during the prior 4 week supplementation period. This was diarized in the GI symptom and training diary.
Post-race
Blood samples were collected immediately post-race for later analysis. Participants were then asked to complete a more detailed questionnaire (adapted from Pfeiffer et al. 2012) to assess any specific symptoms of GI discomfort, including: bloating, flatulence, stitch, belching, nausea, urge to vomit, urge to defecate, and stomach cramps. These were scored on a 10-point scale (0 = no pain and 10 = worst possible pain) with a score > 4 being regarded as moderate. To ensure understanding, specific symptoms were explained and described to participants. This same scale was used in the daily GI symptom diary used during the supplementation period.
Blood analysis
Blood samples were collected into vacutainers containing EDTA, lithium heparin, and serum separation tubes. From whole blood samples, duplicate measures of haematocrit (Hawksley micro-haematocrit reader, Sussex, UK) and haemoglobin (Haemocue, Sussex, UK) were taken. Serum samples were allowed to clot for 1 h at room temperature, while EDTA and lithium heparin samples were immediately stored on ice, following which all samples were centrifuged for 15 min at 1500 RCF at 4 °C. Serum and plasma were manually extracted and stored at − 80 °C until required for analysis. Samples were analysed for plasma glucose, intestinal-fatty acid binding protein (I-FABP), sCD14, interleukin-6 (IL-6), IL-8, IL-10, and serum cortisol. Post-race sample concentrations were corrected for plasma volume changes as described by Dill and Costill (1974).
Intestinal permeability was assessed by analysing serum samples using a previously published protocol (Fleming et al. 1996), with the modification of using rhamnose instead of mannitol as the monosaccharide probe. Briefly, at baseline, a 50 mL sugar probe solution (5 g lactulose, 2 g rhamnose) was consumed and the ratio of the sugars was measured from serum samples 60 min after ingestion. A second identical probe was consumed immediately post-race and serum samples taken after 60 min for LR assessment. Pilot testing within our laboratory showed that after consuming the LR probe, serum lactulose and rhamnose concentrations had returned to baseline 7 h post ingestion and that a second LR probe at this time was able to detect post-exercise LR increase relative to a morning resting sample, which was also demonstrated on race day (Fig. 1b, c).
Concentrations of I-FABP from EDTA plasma were determined using an ELISA (Hycult Biotechnology, Uden, The Netherlands; detection window 47–5000 pg mL−1) according to the manufacturer’s instructions. The coefficient of variation (CV) was 8.0% for between-sample duplicates. Plasma sCD14 was measured with a commercial enzyme-linked immunosorbent assay kit (R&D Systems, Inc., Minneapolis, Minnesota, USA) according to the manufacturer’s instructions, with a CV of 5.9%. Cytokine concentrations were measured using cytometric bead array (CBA, BD Biosciences, San Diego, USA) for the cytokines IL-6, IL-8, and IL-10 using the manufacturer’s instructions with three bead populations with distinct fluorescence intensities coated with capture antibodies specific for IL-6, IL-8, and IL-10 proteins with analysis on a BD Accuri flow cytometer (Becton–Dickinson, Franklin Lakes, NJ, USA). Following acquisition of sample data using the flow cytometer, the sample results were generated in graphical and tabular format using the BD CBA Analysis Software. The combined coefficients of variation were 9.7%. Serum cortisol was measured using an ELISA kit according to the manufacturer’s instructions (Elecsys Cortisol assay, Cobas-Roche, UK), with a CV of 2.9%.
Statistical analysis
Descriptive statistics were produced for all data sets to check for normal distribution indicated by Shapiro–Wilk test (accepted if p > 0.05). A two-factor mixed measures ANOVA was used to examine differences in LR, I-FABP, sCD14, cytokines, and cortisol with condition (PRO and PLC) and various timepoints as the independent variables. Where significant main effects and interactions were present, pairwise comparisons were performed using the Sidak test method. For physiological and symptomology measures, individual data points were consolidated and averaged for each third of the race distance covered. To evaluate data on GI symptoms, a nonparametric statistical approach was chosen, as scores on GI symptoms were mainly reported on the low end of the scale and not normally distributed. Symptom diary variables were compared with the use of Wilcoxon Signed Rank tests. GI symptom diary data were analysed as the sum of all moderate or worse symptoms (scale of 0–84; a maximum of 6 each day) and the total number of days in which any GI symptoms ≥ 4 were reported. Symptom scores during the race were compared with the use of Mann–Whitney nonparametric U test for independent data. Spearman rank-order correlation was used to analyse the relationship between GI symptoms, with post-exercise I-FABP, inflammatory cytokines, and sCD14 concentrations. All normally distributed data are presented as mean ± standard deviation (SD). Data not normally distributed are reported as median and range. p < 0.05 was considered statistically significant. Statistical analysis was conducted using the Statistical Package for the Social Sciences software programme (SPSS, version 23) and Prism statistical software (GraphPad Prism, version.7.0c, La Jolla, CA, USA).