Endurance Trial: ketone and Carbohydrate Supplementation

Design rationalePublished trials of exogenous ketosis in prolonged cycling have used intermittent load,which oscillates intensity and, with it, substrate preference. That format does notfavour expression of the proposed mechanism, whereby ketosis suppresses glycolysis,raises fat oxidation and spares glycogen for a decisive final block. Constant prolongedload was therefore chosen as the more adequate format to test it.Those trials also compare two arms against a carbohydrate background present in everycondition, without a carbohydrate‐free control, which does not separate the contributionof carbohydrate from that of added ketone. The three arms used here resolve this: theplacebo‐to‐carbohydrate contrast measures the contribution of carbohydrate, and thecarbohydrate to carbohydrate‐plus‐ketone contrast measures the contribution of ketoneadded 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 inflammatoryresponse intensify, allowing examination of whether ketosis acts beyond energy provision,as a signalling metabolite on the kynurenine pathway.Pre‐experimental visitBody composition is assessed by dual‐energy X‐ray absorptiometry on a Lunar GE MedicalSystems Prodigy Advance scanner with Encore software version 13.6, calibrated by the sametechnician according to manufacturer recommendations. Lean mass, fat mass, body fatpercentage and bone mineral density are recorded. This assessment is performed once andcharacterizes the sample rather than serving as a study outcome, since it precedes anysupplementation and is not repeated within conditions.The maximal incremental test follows, on a cycle ergometer, with a three‐minute warm‐upat 100 W and increments of 25 W every three minutes until exhaustion. Maximal oxygenuptake is determined by indirect calorimetry on a COSMED Quark CPET analyser, maximalpower output by a Saris power meter with Golden Cheetah software, and heart rate by aPolar chest strap. Perceived exertion is rated on the Borg 0 to 10 scale. At eachincrement, 25 microlitres of capillary blood are sampled from the earlobe for lactatedetermination on a Yellow Springs YSI analyser, from which the anaerobic threshold isderived. Together with maximal power output and maximal oxygen uptake, the thresholdinforms individual load prescription for the experimental sessions.Standardization before each experimental sessionParticipants maintain their habitual dietary pattern and their habitual training volume,intensity and frequency throughout the experimental period. In the three days precedingeach visit, caffeine, non‐steroidal anti‐inflammatory drugs and any medication affectingintestinal transit are suspended. In the week preceding each experimental session,intense effort and systematic training are prohibited, to avoid residual fatigue ormuscle inflammation; adherence is verified by direct contact and by inspection ofindividual training records.Gut training is prescribed in that same week, with progressive carbohydrate volume anddose taken during the participant's own training rides, adapted from the protocol ofCosta et al. (2017), to increase gastrointestinal tolerance to the carbohydrate load ofthe experimental session. Supplements for the following week of gut training are handedto the participant before discharge from each session.Hydration follows 5 mL/kg of cold water on arrival at the laboratory, with ad libitumreplacement throughout the session.Experimental session logisticsEach session runs on a fixed schedule of approximately seven and a half hours fromarrival to discharge.Venous blood is drawn from the antecubital vein into serum and EDTA tubes. Peripheralblood mononuclear cells are isolated from the EDTA tubes by Ficoll‐Hypaque gradient at400 g for 40 minutes. Serum is obtained after 60 minutes at room temperature followed bycentrifugation 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 afterexercise for on‐site determination of lactate, glucose and beta‐hydroxybutyrate on aFreestyle Optium meter, which also serves to verify that the target ketosis was reachedin the CHO‐KET condition. Perceived exertion is rated on the Borg 0 to 10 scale atpredefined moments. Four five‐minute gas exchange windows are recorded during thefixed‐intensity phase, with the mask fitted continuously during the maximal effort block.Before exercise, the cycle ergometer is adjusted to the participant's measurements andthe heart rate monitor is fitted. Gas exchange is recorded across four fixed windows persession: 15 minutes covering rest, warm‐up and exercise onset; 5 minutes at two pointsduring the fixed‐intensity phase; and 30 minutes from 5 minutes before the end of thefixed‐intensity phase through the maximal effort block and the cool‐down. After thecool‐down, the participant completes the post‐exercise cognitive battery, showers, and isweighed. The final venous draw follows the same protocol as baseline.