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.

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