Effects of Acute Exercise in Fasting and Fed States in Individuals With Different Aerobic Fitness Levels

This experimental study uses a mixed design incorporating both between- and within-subject comparisons to investigate the influence of aerobic fitness level, feeding status, and acute exercise on substrate oxidation and peripheral blood mononuclear cell (PBMC) mitochondrial function. Between-group comparisons will examine differences between individuals with high and low aerobic fitness under standardized fasted conditions, whereas within-subject comparisons of fasted and fed conditions will be performed in the high-fitness group. In addition, the repeatability of substrate oxidation measurements will be assessed in the high-fitness group.

A total of 30 healthy men aged 18-25 years with a body mass index (BMI) of 18.5-24.9 kg·m-² will be recruited. Participants will be classified into two groups according to maximal oxygen uptake (V̇O₂max). The high-fitness group will comprise 15 endurance-trained individuals with a V̇O₂max >55 mL·kg-¹·min-¹, whereas the low-fitness group will comprise 15 sedentary or physically inactive individuals with a V̇O₂max <45 mL·kg-¹·min-¹.

During the first laboratory visit, participants in both groups will undergo body composition assessment using dual-energy X-ray absorptiometry (DXA) and complete an incremental cycling test to determine V̇O₂max. Following sufficient recovery, participants will complete a 20-min familiarization cycling session at an intensity corresponding to 65% of V̇O₂max.

Participants in the high-fitness group will complete four laboratory visits in total. During the second and third visits, participants will arrive at the laboratory following an overnight fast. Resting metabolic rate (RMR) will be assessed, after which participants will complete 30 min of cycling exercise at 65% of V̇O₂max. These visits will be performed under identical standardized conditions to determine the repeatability of substrate oxidation measurements.

During the fourth visit, participants in the high-fitness group will arrive at the laboratory following an approximately 12-h overnight fast. RMR will first be assessed, followed by the collection of a venous blood sample immediately before exercise. Participants will then complete 30 min of cycling at 65% of V̇O₂max, and a second venous blood sample will be collected immediately after exercise. These samples will be used to assess exercise-induced changes in PBMC mitochondrial function under fasted conditions.

One hour after completing the fasted exercise trial, participants will consume an ad libitum meal consisting of pasta and meatballs. One hour after completion of the meal, RMR assessment and the 30-min cycling exercise trial at 65% of V̇O₂max will be repeated under fed conditions. No blood samples will be collected during the fed condition. Accordingly, comparisons between fasted and fed conditions will be restricted to outcomes related to whole-body substrate metabolism.

Participants in the low-fitness group will complete two laboratory visits. The first visit will include DXA assessment, determination of V̇O₂max, and the familiarization cycling session, as described above. During the second visit, participants will complete the fasted experimental condition. Following an approximately 12-h overnight fast, RMR will be assessed and a venous blood sample will be collected immediately before exercise. Participants will then complete 30 min of cycling at 65% of V̇O₂max, followed immediately by collection of a second venous blood sample. The low-fitness group will not complete the repeatability trials or the fed condition. Therefore, between-group comparisons of substrate oxidation and PBMC mitochondrial function will be performed using data obtained under the standardized fasted condition.

Respiratory gas exchange will be continuously measured during resting and exercise assessments to determine oxygen consumption (V̇O₂) and carbon dioxide production (V̇CO₂). These measurements will be used to calculate rates of fat and carbohydrate oxidation. Rating of perceived exertion (RPE) will be recorded every 5 min during exercise using the Borg 6-20 scale.

PBMCs will be isolated from venous blood samples collected immediately before and after the fasted exercise test. Mitochondrial oxygen consumption rate and proton efflux rate will be measured using a Seahorse XFe96 extracellular flux analyzer. These measurements will be used to evaluate exercise-induced changes in PBMC mitochondrial function and their relationship with whole-body fat and carbohydrate oxidation during exercise.

Participants will also complete a 24-hour dietary intake record before the main experimental visit.

Inclusion criteria

• Healthy male volunteers aged 18-25 years
• Body mass index between 18.5 and 24.9 kg/m²
• Residing on the Hacettepe University Beytepe Campus
• Eligibility for one of the following aerobic fitness groups

• High aerobic fitness group: maximal oxygen uptake (VO2max) greater than 55 mL/kg/min and regular participation in endurance training.
• Low aerobic fitness group: VO2max lower than 45 mL/kg/min and sedentary or not regularly physically active

Exclusion criteria

• Current smoking
• Regular or excessive alcohol consumption
• Any musculoskeletal condition that may limit or prevent exercise
• Allergy to any of the foods provided as part of the study meal
• Currently following a weight-loss or other restrictive diet