FueL Use During eXercise in Males and Females

Sex differences in substrate utilization during fasted endurance exercise are well established, with females generally demonstrating greater reliance on fat oxidation and lower carbohydrate utilization than males. However, exercise is frequently performed following nutrient ingestion, and it remains unclear whether sex differences in substrate utilization persist when carbohydrate or protein is consumed before exercise.

This randomized, counterbalanced crossover study will compare three pre-exercise nutritional conditions: carbohydrate (CHO), protein (PRO), and fasted/placebo (FAST). Participants will complete each condition separated by a minimum three-day washout. Following an overnight fast, participants will consume the assigned beverage and rest for 30 minutes before completing 90 minutes of cycling at 65% of VO2peak. This will be followed by a two-minute rest and a cycling time-to-task-failure test at 85% of maximal work rate.

The primary objective is to determine whether pre-exercise nutrition modifies sex differences in whole-body carbohydrate and fat oxidation and exogenous substrate oxidation during exercise. A secondary objective is to determine the effects of pre-exercise nutrition and sex on subsequent exercise performance.

• Pre-exercise carbohydrate beverage (Dietary supplement): A carbohydrate beverage providing 1 g glucose/kg body mass, enriched with 1 mg/kg body mass \[U-13C6\]-D-glucose for assessment of exogenous carbohydrate oxidation, with calorie-free flavouring. The beverage will be consumed within 5 minutes, 30 minutes before exercise.
• Pre-exercise protein beverage (Dietary supplement): A protein-based beverage providing 0.45 g protein/kg body mass and supplemented with 0.24 g fat/kg body mass to match the energy content of the carbohydrate condition. The beverage is enriched with L-\[1-13C\]leucine and flavour-matched to the carbohydrate beverage. It will be consumed within 5 minutes, 30 minutes before exercise.
• Fasted control beverage (Other): A non-caloric beverage consisting of water and sugar-free flavouring, flavour-matched to the carbohydrate beverage. The beverage will be consumed within 5 minutes following an overnight fast, 30 minutes before exercise.

Primary outcomes

• Whole-body carbohydrate oxidation [During the 90-minute exercise bout]: Whole-body carbohydrate oxidation will be calculated from oxygen consumption (VO2) and carbon dioxide production (VCO2) measured by indirect calorimetry during 90 minutes of moderate-intensity cycling. Carbohydrate oxidation will be compared between the carbohydrate, protein, and fasted conditions and between females and males.
• Whole-body fat oxidation [During the 90-min exercise bout]: Whole-body fat oxidation will be calculated from VO2 and VCO2 measured by indirect calorimetry during 90 minutes of moderate-intensity cycling. Fat oxidation will be compared between the carbohydrate, protein, and fasted conditions and between females and males.
• Exogenous glucose oxidation [During the 90-min exercise bout]: Exogenous glucose oxidation will be determined during the carbohydrate condition using breath 13CO2 enrichment following ingestion of \[U-13C6\]-D-glucose. Breath samples will be analyzed by isotope ratio mass spectrometry, and exogenous glucose oxidation will be calculated using 13CO2 enrichment and carbon dioxide production.
• Exogenous leucine oxidation [During the 90-min exercise bout]: Exogenous leucine oxidation will be determined during the protein condition using breath ¹³CO₂ enrichment following ingestion of L-\[1-¹³C\]leucine. Breath samples will be analyzed by isotope ratio mass spectrometry, and exogenous leucine oxidation will be calculated using breath ¹³CO₂ enrichment and carbon dioxide production.

Secondary outcomes

• Time to task failure [Immediately following the 90-min exercise bout]: Exercise performance will be assessed as time to volitional exhaustion during a cycling time-to-task-failure test performed at 85% of maximal work rate following the 90-minute moderate-intensity cycling bout.

Sex: All, ages: 18 Years to 35 Years, accepts healthy volunteers

Inclusion Criteria:

* Participating in ≥150 minutes of moderate-to-vigorous physical activity per week
* VO₂peak classified as "good" or above based on American College of Sports Medicine normative values: >35.0 mL/kg/min for females and >39.0 mL/kg/min for males
* Body mass index (BMI) between 18 and 27 kg/m²
* Participating in 3-5 structured exercise sessions per week
* Weight stable, defined as within ±2 kg for at least 6 months
* Non-smoker
* Females must be eumenorrheic

Exclusion Criteria:

* Polycystic ovarian syndrome (PCOS) or endometriosis Current use of oral contraceptives or oral contraceptive use within the previous 3 months Diagnosed cardiovascular or metabolic disease Hypergonadism or hypogonadism Use of medications for management of blood glucose or lipid metabolism Pregnant or <12 months postpartum Lactating or breastfeeding within 3 months of study commencement Menopausal Recreational tobacco smoking Inability to complete the study exercise protocols or comply with pre-trial dietary or physical activity controls Use of medications that may affect substrate metabolism, including corticosteroids or non-steroidal anti-inflammatory drugs (NSAIDs) Currently following a low-carbohydrate diet, such as a ketogenic or Atkins diet

• Jenna Gillen, PhD, Principal investigator, University of Toronto