Endurance Trial: Ketone and Carbohydrate Supplementation

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 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 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

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.

Cognitive flexibility is assessed with the Trail Making Test, parts A and B, from the PEBL battery. In part A the participant selects numbered circles from 1 to 26 in ascending order as fast as possible; in part B the participant alternates between numbers and letters. The difference between the completion times of part B and part A is reported, with lower values indicating greater cognitive flexibility.

Unit: milliseconds

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 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

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 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

Mood is assessed with the Brunel Mood Scale (BRUMS), the Brazilian version of a shortened Profile of Mood States. The instrument comprises 24 single-word mood descriptors distributed across six subscales of four items each: tension, depression, anger, vigour, fatigue and confusion. Each item is rated on a 5-point Likert scale from 0 (not at all) to 4 (extremely), so that each subscale score ranges from 0 to 16, with higher scores indicating greater intensity of that mood dimension. The fatigue and vigour subscales are the variables of interest. Unit: score on a scale

Dietary intake is assessed by a 24-hour dietary recall conducted in person by a nutritionist using the five-step multiple-pass method, referring to the day preceding each visit. Participants are instructed to maintain their habitual diet and physical activity throughout the experimental period, and protein, carbohydrate and micronutrient supplements are quantified together with food. Records are analysed using the Brazilian food composition tables TBCA-USP and TACO-UNICAMP and, when an item is not listed, the United States Department of Agriculture National Nutrient Database for Standard Reference. Total daily energy intake is reported. Unit: kilocalories per day.

Total RNA is extracted from peripheral blood mononuclear cells and reverse transcribed. Gene expression of targets related to the kynurenine pathway, endoplasmic reticulum stress, autophagy and oxidative stress is quantified by real-time quantitative PCR using SYBR Green chemistry, and reported as relative expression by the 2^-ddCt method against a reference gene.

Unit: relative expression, 2^-ddCt

Serum concentrations of kynurenine pathway metabolites, including kynurenine, kynurenic acid, 3-hydroxykynurenine and quinolinic acid, are quantified by high-performance liquid chromatography coupled to tandem mass spectrometry.

Unit: micromoles per litre

Circulating concentration of Gram-negative bacterial endotoxin is measured using a chromogenic Limulus amebocyte lysate assay (HIT302, Hycult Biotech, Uden, Netherlands), in duplicate, with all samples from a given participant assayed on the same plate.

Unit: endotoxin units per millilitre

Serum concentration of endotoxin core antibody is quantified by enzyme-linked immunosorbent assay (HK504, Hycult Biotech, Uden, Netherlands), in duplicate, with all samples from a given participant assayed on the same plate.

Unit: median metric units per millilitre

Serum cortisol concentration is quantified by enzyme-linked immunosorbent assay, in duplicate, with all samples from a given participant assayed on the same plate. Unit: micrograms per decilitre

Concentrations of interleukin-6, interleukin-4, nuclear factor kappa-B, tumour necrosis factor, interleukin-1 beta and interleukin-10 are quantified in serum, in the culture medium of peripheral blood mononuclear, and cells co-cultured with SH-SY5Y neuronal cells. Unit: picograms per millilitre.

Subcutaneous fat is estimated from skinfold thickness measured with a Lange skinfold caliper by the same trained assessor at every visit. At the experimental sessions the measurement is performed between the first and second supplement doses, together with the 24-hour dietary recall. The sum of skinfolds is reported. Unit: millimetres

Body circumferences are measured with an inelastic anthropometric tape by the same trained assessor, following the same schedule as the skinfold measurements. Unlike skinfold thickness, which estimates subcutaneous adiposity, circumferences characterize regional body dimensions and are reported individually by site. Unit: centimetres

Body mass is measured on a calibrated digital scale with the participant wearing minimal clothing. Unlike the skinfold and circumference measurements, which are recorded once per visit, body mass is recorded twice at each experimental session: before exercise, between the first and second supplement doses, and again after exercise, following a shower and before the final venous blood draw. The difference between the two measurements is used to estimate fluid loss during exercise. Unit: kilograms

Carbohydrate, protein and fat intake as well as micronutrient intake are derived from the same 24-hour dietary recall and analysed with the same food composition tables described for daily energy intake, and are reported in grams per day. Unit: grams per day.

Pulmonary gas exchange is measured breath-by-breath on a COSMED Quark CPET analyser with Omnia software, across four fixed windows per session: a 15-minute window covering seated rest, warm-up and the onset of exercise; two 5-minute windows during the fixed-intensity phase; and a 30-minute window beginning 5 minutes before the end of the fixed-intensity phase and running through the 20-minute maximal effort block and the 5-minute cool-down. The respiratory exchange ratio is calculated as carbon dioxide production divided by oxygen uptake and reported as the mean of each window. A lower ratio indicates a greater contribution of fat oxidation to energy provision. Unit: ratio.

Fat and carbohydrate oxidation rates are derived from oxygen uptake and carbon dioxide production measured breath-by-breath on a COSMED Quark CPET analyser, using stoichiometric equations, and are reported separately in the same unit as the mean of each of the four measurement windows described for the respiratory exchange ratio. This outcome tests whether exogenous ketosis shifts substrate preference toward fat oxidation during prolonged constant-load exercise, and characterizes the substrate mix sustaining the effort in each condition. Unit: grams per minute.

Oxygen uptake is measured breath-by-breath on a COSMED Quark CPET analyser, normalized to body mass and reported as the mean of each of the four measurement windows. Because external load during the fixed-intensity phase is individually prescribed and identical across conditions, differences in oxygen uptake between conditions reflect differences in exercise economy. Unit: millilitres per kilogram per minute.

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

Unit: kilojoules