Endurance Trial: Ketone and Carbohydrate Supplementation

Distance covered during the final 20 minutes of maximal effort

Physical performance is assessed as the distance covered during the final 20-minute maximal effort block of each experimental session. Each session begins with 5 minutes of warm-up at 100 W, followed by 155 minutes at 55% of maximal power output (Wmax) with the cycle ergometer set in hyperbolic mode, and ends with 20 minutes of maximal self-paced effort in linear mode. Load is individually prescribed from the maximal incremental test performed at Visit 1. Participants ride the bicycle they are accustomed to, mounted on an electromagnetically braked cycle ergometer, and distance is recorded by the ergometer software. Unit: kilometres

Visuospatial working memory: Corsi block-tapping span

Visuospatial working memory is assessed with the Corsi block-tapping task from the Psychology Experiment Building Language (PEBL) battery. Sequences of illuminated squares are presented, starting with two blocks and increasing by one every two attempts, until two consecutive errors occur. The span is the length of the longest sequence correctly reproduced; higher values indicate better working memory. Participants are familiarized with the task at Visit 1, and a brief rehearsal is performed immediately before testing at each experimental session. Unit: number of blocks

Inhibitory control: accuracy on No-Go trials

Inhibitory control is assessed with the Go/No-Go task from the PEBL battery. The letters P or R replace a blue star on screen, with 10 practice trials followed by 160 stimuli per block in an 80:20 Go to No-Go ratio, a 500-millisecond stimulus duration and a 1500-millisecond inter-stimulus interval. Accuracy on No-Go trials is reported as the proportion of correctly withheld responses, ranging from 0 to 1, with higher values indicating better inhibitory control. Unit: proportion of correct responses

Processing speed: reaction time on Go trials

Reaction time on correct Go trials of the Go/No-Go task described above is recorded automatically by the PEBL software and reported as the mean latency in milliseconds. Lower values indicate faster processing speed.

Gastrointestinal symptom severity

A 10-point Likert-type scale, adapted into a 20 cm visual analogue scale, is used, where 0 indicates absence of symptoms and 10 indicates extreme symptoms, so that higher scores indicate worse tolerance. The questionnaire comprises three sections: upper abdominal symptoms (reflux, abdominal distension, nausea, vomiting); lower abdominal symptoms (cramps, flatulence, abdominal pain, diarrhoea); and systemic symptoms (dizziness, headache, muscle cramps). Unit: score on a 0 to 10 scale

Serum intestinal fatty acid-binding protein concentration

Serum concentration of intestinal fatty acid-binding protein (I-FABP) is quantified by enzyme-linked immunosorbent assay (HK406, Hycult Biotech, Uden, Netherlands). Samples are analysed in duplicate according to the manufacturer's instructions on the same day, with standards and controls on each plate, and all samples from a given participant are assayed on the same plate to minimize inter-assay variability. Unit: picograms per millilitre

Protein expression of kynurenine pathway, endoplasmic reticulum stress, autophagy and oxidative stress markers in PBMCs

Peripheral blood mononuclear cells are isolated from whole blood by Ficoll-Hypaque density-gradient centrifugation. Total protein is extracted with RIPA buffer containing protease inhibitors and quantified by the bicinchoninic acid assay. Protein expression of enzymes of the kynurenine pathway and of markers of endoplasmic reticulum stress, autophagic flux and oxidative stress is determined by Western blot after SDS-PAGE separation, transfer to nitrocellulose membrane, overnight incubation with specific primary antibodies, incubation with a peroxidase-conjugated secondary antibody, chemiluminescent detection and densitometric analysis. Unit: arbitrary units, normalized to loading control

Sleep difficulty score on the Athlete Sleep Screening Questionnaire

Sleep is assessed with the Athlete Sleep Screening Questionnaire (ASSQ), validated for Brazilian athletes. The instrument comprises 15 items covering sleep quantity, sleep quality, insomnia and chronotype; only the first seven items are used, as these generate the sleep difficulty score (SDS). The SDS ranges from 0 to 17, with higher scores indicating greater sleep difficulty, and classifies athletes into four categories of clinical sleep problems: none (0 to 4), mild (5 to 7), moderate (8 to 10) and severe (11 to 17). The questionnaire refers to the 7 days preceding its administration. Because the three experimental sessions are separated by a one-week washout, each administration characterizes sleep during the week that precedes the corresponding condition. Unit: score on a scale

Total work produced during the final 20 minutes of maximal effort

Total mechanical work produced during the final 20-minute maximal effort block, calculated from power output recorded continuously by the cycle ergometer. Unit: kilojoules