Endurance Trial: Ketone and Carbohydrate Supplementation

Double-Blind Randomized Crossover Trial Examining the Effects of Ketone and Carbohydrate Supplementation on Molecular Biomarkers, Cognitive Function, and Physical Performance in Healthy Endurance Athletes

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.

Inclusion Criteria:

• Amateur male cyclists;
• At least 2 years of cycling experience;
• At least 5 hours of endurance cycling training per week;
• VO2max of at least 50 mL/kg/min.

Exclusion Criteria:

• Confirmed diagnosis of COVID-19 by PCR or rapid antigen test within the last 12 months;
• Symptoms compatible with COVID-19 within the last six months;
• Smokers;
• Alcoholics;
• Presence of neurological, immune, or muscular diseases;
• Use of psychoactive medications in the last year;
• Use of any acute medication prior three days each visit that possible could affect the experiment such as stool-altering medications and non-steroidal anti-inflammatory drugs;
• Confirmed gastrointestinal infections, diseases, or disorders (e.g., coeliac disease, inflammatory bowel disease, irritable bowel syndrome, diverticular disease, gastroesophageal reflux disease);
• Medical history of gastrointestinal surgery, or any other self-reported gastrointestinal issues;
• Within the previous three months, if they had consumed substances that could affect gastrointestinal integrity or followed gastrointestinal-targeted dietary regimes;
• Participants with any food allergy should be excluded of this study.