This randomized, double-blind, crossover study investigates the effects of exogenous ketone supplementation on physical and cognitive performance in amateur endurance athletes during exhaustive exercise. Participants will complete three 3-hour cycling sessions under different nutritional conditions: carbohydrate alone (CHO), carbohydrate plus ketone (CHO-KET), and placebo (PLA). The study will assess cognitive function through validated computerized tests, physical performance via distance covered, and metabolic responses through blood biomarkers. Additionally, peripheral blood mononuclear cells (PBMCs) will be collected and cultured with LPS or not. Other PBMCs co-cultured with neuronal cells (SH-SY5Y) to investigate neuro-immune interactions. The primary hypothesis is that ketone supplementation will preserve cognitive function and enhance physical performance during prolonged exhaustive exercise compared to carbohydrate supplementation alone, potentially through anti-inflammatory mechanisms and alternative energy substrate provision.
Design rationale Published trials of exogenous ketosis in prolonged cycling have used intermittent load, which oscillates intensity and, with it, substrate preference. That format does not favour expression of the proposed mechanism, whereby ketosis suppresses glycolysis, raises fat oxidation and spares glycogen for a decisive final block. Constant prolonged load was therefore chosen as the more adequate format to test it. Those trials also compare two arms against a carbohydrate background present in every condition, without a carbohydrate-free control, which does not separate the contribution of carbohydrate from that of added ketone. The three arms used here resolve this: the placebo-to-carbohydrate contrast measures the contribution of carbohydrate, and the carbohydrate to carbohydrate-plus-ketone contrast measures the contribution of ketone added on a matched background, added rather than substituted on an isocaloric basis. The three-hour window was chosen because it is where cortisol and the inflammatory response intensify, allowing examination of whether ketosis acts beyond energy provision, as a signalling metabolite on the kynurenine pathway. Pre-experimental visit Body composition is assessed by dual-energy X-ray absorptiometry on a Lunar GE Medical Systems Prodigy Advance scanner with Encore software version 13.6, calibrated by the same technician according to manufacturer recommendations. Lean mass, fat mass, body fat percentage and bone mineral density are recorded. This assessment is performed once and characterizes the sample rather than serving as a study outcome, since it precedes any supplementation and is not repeated within conditions. The maximal incremental test follows, on a cycle ergometer, with a three-minute warm-up at 100 W and increments of 25 W every three minutes until exhaustion. Maximal oxygen uptake is determined by indirect calorimetry on a COSMED Quark CPET analyser, maximal power output by a Saris power meter with Golden Cheetah software, and heart rate by a Polar chest strap. Perceived exertion is rated on the Borg 0 to 10 scale. At each increment, 25 microlitres of capillary blood are sampled from the earlobe for lactate determination on a Yellow Springs YSI analyser, from which the anaerobic threshold is derived. Together with maximal power output and maximal oxygen uptake, the threshold informs individual load prescription for the experimental sessions. Standardization before each experimental session Participants maintain their habitual dietary pattern and their habitual training volume, intensity and frequency throughout the experimental period. In the three days preceding each visit, caffeine, non-steroidal anti-inflammatory drugs and any medication affecting intestinal transit are suspended. In the week preceding each experimental session, intense effort and systematic training are prohibited, to avoid residual fatigue or muscle inflammation; adherence is verified by direct contact and by inspection of individual training records. Gut training is prescribed in that same week, with progressive carbohydrate volume and dose taken during the participant's own training rides, adapted from the protocol of Costa et al. (2017), to increase gastrointestinal tolerance to the carbohydrate load of the experimental session. Supplements for the following week of gut training are handed to the participant before discharge from each session. Hydration follows 5 mL/kg of cold water on arrival at the laboratory, with ad libitum replacement throughout the session. Experimental session logistics Each session runs on a fixed schedule of approximately seven and a half hours from arrival to discharge. Venous blood is drawn from the antecubital vein into serum and EDTA tubes. Peripheral blood mononuclear cells are isolated from the EDTA tubes by Ficoll-Hypaque gradient at 400 g for 40 minutes. Serum is obtained after 60 minutes at room temperature followed by centrifugation at 1300 g for 10 minutes, and stored at minus 80 degrees Celsius. Capillary blood is sampled from the earlobe at defined intervals before, during and after exercise for on-site determination of lactate, glucose and beta-hydroxybutyrate on a Freestyle Optium meter, which also serves to verify that the target ketosis was reached in the CHO-KET condition. Perceived exertion is rated on the Borg 0 to 10 scale at predefined moments. Four five-minute gas exchange windows are recorded during the fixed-intensity phase, with the mask fitted continuously during the maximal effort block. Before exercise, the cycle ergometer is adjusted to the participant's measurements and the heart rate monitor is fitted. Gas exchange is recorded across four fixed windows per session: 15 minutes covering rest, warm-up and exercise onset; 5 minutes at two points during the fixed-intensity phase; and 30 minutes from 5 minutes before the end of the fixed-intensity phase through the maximal effort block and the cool-down. After the cool-down, the participant completes the post-exercise cognitive battery, showers, and is weighed. The final venous draw follows the same protocol as baseline.
Study Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
BASIC_SCIENCE
Masking
QUADRUPLE
Enrollment
12
Participants ingest 210 mL of a \~15% carbohydrate solution, providing 30 g of carbohydrate per dose, every 20 minutes from the start of the 155-minute fixed-intensity phase, with the final dose taken immediately before the start of the 20-minute maximal effort block. No beverage is provided during the maximal effort. The solution combines sucrose and dextrose so that glucose and fructose are absorbed through separate intestinal transporters. All doses are supplied in opaque, coded bottles prepared by a researcher not involved in the assessments, so that treatments cannot be identified visually.
Participants ingest (R)-1,3-butanediol at 1.0 g/kg body mass 45 minutes before the start of the cycling protocol and a further 0.5 g/kg 5 minutes before the start, for a total dose of 1.5 g/kg body mass. The dose was selected to sustain blood beta-hydroxybutyrate at approximately 2 mmol/L throughout the session, a range compatible with nutritional ketosis and without risk to participants. Doses are supplied in opaque, coded bottles prepared by a researcher not involved in the assessments.
Participants ingest a non-caloric placebo beverage at 45 and 5 minutes before the start of the cycling protocol, at the same time points at which the (R)-1,3-butanediol is administered in the CHO-KET arm. The placebo is matched to the ketone beverage for volume, appearance and flavour, and provides no energy. Doses are supplied in opaque, coded bottles prepared by a researcher not involved in the assessments.
Participants ingest 210 mL of a non-caloric placebo beverage every 20 minutes from the start of the 155-minute fixed-intensity phase, with the final dose taken immediately before the start of the 20-minute maximal effort block. No beverage is provided during the maximal effort. The placebo is water with the same flavouring used in the carbohydrate beverage, matched for volume, appearance and ingestion schedule, and provides no energy. Doses are supplied in opaque, coded bottles prepared by a researcher not involved in the assessments.
On the evening before each experimental session, participants consume a standardized meal providing 1.5 g of carbohydrate per kg of body mass. The same meal is provided before all three experimental sessions and is followed by an overnight fast of 8 to 12 hours.
Each experimental session comprises 5 minutes of warm-up at 100 W, 155 minutes at 55% of maximal power output (Wmax) with the cycle ergometer set in hyperbolic mode, and a final 20 minutes of maximal self-paced effort in linear mode, followed by 5 minutes of low-load cool-down. The same protocol is applied in all three conditions, with load individually prescribed from the maximal incremental test performed at the pre-experimental visit.
On the morning of each experimental session, after the baseline venous blood draw and following an overnight fast of 8 to 12 hours, participants consume a standardized breakfast providing 1.5 g of carbohydrate per kg of body mass, 40 g of protein and 7 g of fat. Participants are instructed to consume the entire meal. The same breakfast is provided before all three experimental sessions.
School of Physical Education and Sport of Ribeirão Preto - University of São Paulo
Ribeirão Preto, São Paulo, Brazil
RECRUITINGDistance 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
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): final 20 minutes of exercise.
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
Time frame: Visit 1 (week 0), familiarization session. Visits 2, 3 and 4 (weeks 3, 4 and 5): 15 minutes after the standardized breakfast, before the first supplement dose; and 5 minutes after the end of exercise.
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
Time frame: Visit 1 (week 0), familiarization session. Visits 2, 3 and 4 (weeks 3, 4 and 5): 15 minutes after the standardized breakfast, before the first supplement dose; and 5 minutes after the end of exercise.
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.
Time frame: Visit 1 (week 0), familiarization session. Visits 2, 3 and 4 (weeks 3, 4 and 5): 15 minutes after the standardized breakfast, before the first supplement dose; and 5 minutes after the end of exercise.
Cognitive flexibility: Trail Making Test B minus A completion time
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
Time frame: Visit 1 (week 0), familiarization session. Visits 2, 3 and 4 (weeks 3, 4 and 5): 15 minutes after the standardized breakfast, before the first supplement dose; and 5 minutes after the end of exercise.
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
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): immediately before breakfast; immediately before exercise; 40, 100 and 160 minutes after exercise onset; immediately after exercise; and 60 minutes after exercise.
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
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): baseline on arrival after an 8 to 12 hour overnight fast, and 60 minutes after the end of exercise.
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
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): baseline on arrival after an 8 to 12 hour overnight fasting, and 60 minutes after the end of exercise.
Sleep difficulty score on the Athlete Sleep Screening Questionnaire
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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
Time frame: Sleep during the 7 days preceding each assessment. Assessed at Visit 1 (week 0) and at Visits 2, 3 and 4 (weeks 3, 4 and 5). At the experimental sessions the questionnaire is administered immediately after the standardized breakfast.
Fatigue and vigour subscale scores on the Brunel Mood 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
Time frame: Visit 1 (week 0). Visits 2, 3 and 4 (weeks 3, 4 and 5): immediately after the baseline cognitive battery, 45 minutes before exercise; and immediately after the post-exercise cognitive battery, 40 minutes after exercise.
Daily energy intake
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.
Time frame: Visit 1 (week 0): after DEXA. Visits 2, 3 and 4 (weeks 3, 4 and 5): 45 to 5 minutes before the start of exercise, between the first and second supplement doses.
Gene expression of kynurenine pathway, endoplasmic reticulum stress, autophagy and oxidative stress targets in PBMCs
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
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): baseline on arrival after an 8 to 12 hour overnight fast, and 60 minutes after the end of exercise.
Serum concentration of kynurenine pathway metabolites
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
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): baseline on arrival after an 8 to 12 hour overnight fast, and 60 minutes after the end of exercise.
Serum endotoxin concentration
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
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): baseline on arrival after an 8 to 12 hour overnight fast, and 60 minutes after the end of exercise.
Serum endotoxin core antibody concentration
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
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): baseline on arrival after an 8 to 12 hour overnight fast, and 60 minutes after the end of exercise.
Serum cortisol concentration
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
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): baseline on arrival after an 8 to 12 hour overnight fast, and 60 minutes after the end of exercise.
Concentration of inflammatory cytokines in serum and cell culture medium
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.
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5). Serum: baseline after an overnight fast and 60 minutes after exercise. Culture medium: before and after 24 hours of incubation of cells isolated at those two time points.
skinfold thicknesses
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
Time frame: Visit 1 (week 0): after DEXA, before maximum incremental exercise. Visits 2, 3 and 4 (weeks 3, 4 and 5): 45 to 5 minutes before the start of exercise, between the first and second supplement doses.
Body circumferences
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
Time frame: Visit 1 (week 0): after DEXA, before maximum incremental exercise. Visits 2, 3 and 4 (weeks 3, 4 and 5): 45 to 5 minutes before the start of exercise, between the first and second supplement doses.
Body mass
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
Time frame: Visit 1 (week 0). Visits 2, 3 and 4 (weeks 3, 4 and 5): 45 to 5 minutes before the start of exercise, and 45 minutes after the end of exercise.
Daily nutrient intake
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.
Time frame: Visit 1 (week 0): after DEXA. Visits 2, 3 and 4 (weeks 3, 4 and 5): 45 to 5 minutes before the start of exercise, between the first and second supplement doses.
Respiratory exchange ratio during exercise
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.
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): 15-minute window covering rest, warm-up and exercise onset; 5-minute windows at minutes 60 and 120; and a 30-minute window from minute 155, covering the last 5 minutes of fixed load, maximal effort and cool-down.
Substrate oxidation rate during exercise
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.
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): 15-minute window covering rest, warm-up and exercise onset; 5-minute windows at minutes 60 and 120; and a 30-minute window from minute 155, covering the last 5 minutes of fixed load, maximal effort and cool-down.
Oxygen uptake during exercise
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.
Time frame: Visits 2, 3 and 4 (weeks 3, 4 and 5): 15-minute window covering rest, warm-up and exercise onset; 5-minute windows at minutes 60 and 120; and a 30-minute window from minute 155, covering the last 5 minutes of fixed load, maximal effort and cool-down.