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

The Effect of Acute Exercise in the Fasted and Fed States on Peripheral Blood Mononuclear Cells and Metabolic Fuel Utilization in Individuals With Different Aerobic Fitness Levels

The aim of this study is to investigate the effects of aerobic fitness level, feeding status, and acute exercise on whole-body metabolic fuel utilization and mitochondrial function in peripheral blood mononuclear cells (PBMCs) in healthy young men.

The main questions it aims to answer are:

1. Do individuals with high and low aerobic fitness levels differ in fat and carbohydrate oxidation at rest and during exercise following an overnight fast, and do their PBMC mitochondrial responses to acute exercise differ?
2. Does feeding alter fat and carbohydrate oxidation at rest and during exercise compared with the fasted state in individuals with high aerobic fitness?
3. Are PBMC mitochondrial respiration parameters and their exercise-induced changes associated with fat and carbohydrate oxidation during fasted exercise?

Study Overview

• Status: Not yet recruiting
• Conditions: Physical Fitness; Energy Metabolism; Mitochondria; Mitochondrial Function, Bioenergetics
• Intervention / Treatment: Behavioral: Acute Moderate-Intensity Cycling Exercise; Other: Ad Libitum Mixed Meal

Detailed Description

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.

• Study Type: Interventional
• Enrollment (Estimated): 30
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Burcu ÖZALP, MSc Student; Phone Number: +905539286638; Email: burcu.ozalp@hacettepe.edu.tr
• Study Contact Backup: Name: Süleyman BULUT, PhD; Phone Number: 137 +903122976890; Email: slmbulut@hacettepe.edu.tr

Study Locations

• Turkey (Türkiye)
  • Ankara
    • Ankara, Ankara, Turkey (Türkiye), 06800
      • Hacettepe University, Faculty of Sport Sciences
      • Contact: Burcu ÖZALP, MSc Student; Phone Number: +905539286638; Email: burcu.ozalp@hacettepe.edu.tr
      • Contact: Süleyman BULUT, PhD; Phone Number: 137 +903122976890; Email: slmbulut@hacettepe.edu.tr

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: Yes

Description

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

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Non-Randomized
• Interventional Model: Parallel Assignment
• Masking: None (Open Label)

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: High Aerobic Fitness: Fasted and Fed Conditions; Participants with a VO2max greater than 55 mL/kg/min who regularly perform endurance training will be assigned to this arm. Participants will complete two repeatability visits under overnight-fasted conditions, including resting metabolic rate measurement followed by 30 minutes of cycling at 65% of VO2max. During the main experimental visit, they will complete the same measurements in the fasted state, with venous blood samples collected immediately before and after exercise. They will subsequently consume an ad libitum meal, and one hour later, resting metabolic rate measurement and the cycling exercise test will be repeated in the fed state.	Behavioral: Acute Moderate-Intensity Cycling Exercise; Participants will perform 30 minutes of continuous cycling exercise on a cycle ergometer at an intensity corresponding to 65% of their individual maximal oxygen uptake (VO2max). Both aerobic fitness groups will complete the exercise following an overnight fast. Participants in the high aerobic fitness group will also repeat the exercise one hour after consuming an ad libitum meal. Respiratory gas exchange will be measured throughout exercise to calculate fat and carbohydrate oxidation, and rating of perceived exertion will be recorded every five minutes using the Borg 6-20 scale.; Other: Ad Libitum Mixed Meal; One hour after completing the fasted exercise test, participants in the high aerobic fitness group will be provided with an ad libitum mixed meal consisting of pasta and meatballs and will be allowed to eat until comfortably full. One hour after the meal, resting metabolic rate measurement and the 30-minute cycling exercise test at 65% of VO2max will be repeated under the fed condition. No blood samples will be collected during the fed condition.
Other: Low Aerobic Fitness: Fasted Condition; Participants with a VO2max lower than 45 mL/kg/min who are sedentary or physically inactive will be assigned to this arm. Participants will complete the fasted experimental condition only. Following an overnight fast, resting metabolic rate will be measured and a venous blood sample will be collected immediately before 30 minutes of cycling at 65% of VO2max. A second venous blood sample will be collected immediately after exercise. Participants in this arm will not complete the repeatability visits or the fed experimental condition.	Behavioral: Acute Moderate-Intensity Cycling Exercise; Participants will perform 30 minutes of continuous cycling exercise on a cycle ergometer at an intensity corresponding to 65% of their individual maximal oxygen uptake (VO2max). Both aerobic fitness groups will complete the exercise following an overnight fast. Participants in the high aerobic fitness group will also repeat the exercise one hour after consuming an ad libitum meal. Respiratory gas exchange will be measured throughout exercise to calculate fat and carbohydrate oxidation, and rating of perceived exertion will be recorded every five minutes using the Borg 6-20 scale.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Change in PBMC Mitochondrial Oxygen Consumption Rate From Pre- to Post-Exercise	Mitochondrial oxygen consumption rate (OCR) will be measured in peripheral blood mononuclear cells (PBMCs) using a Seahorse XFe96 extracellular flux analyzer. PBMCs will be isolated from venous blood samples collected immediately before and immediately after the fasted 30-minute submaximal cycling test. Pre- to post-exercise changes in OCR will be evaluated between the high and low aerobic fitness groups and examined in relation to whole-body fat and carbohydrate oxidation during exercise.	Immediately before and immediately after the fasted 30-minute cycling test at 65% of VO2max.
Change in PBMC Proton Efflux Rate From Pre- to Post-Exercise	Proton efflux rate (PER) will be measured in peripheral blood mononuclear cells (PBMCs) using a Seahorse XFe96 extracellular flux analyzer. PBMCs will be isolated from venous blood samples collected immediately before and immediately after the fasted 30-minute submaximal cycling test. Pre- to post-exercise changes in PER will be evaluated between the high and low aerobic fitness groups and examined in relation to whole-body substrate oxidation during exercise.	Immediately before and immediately after the fasted 30-minute cycling test at 65% of VO2max.
Fat Oxidation Rate During Submaximal Exercise	Whole-body fat oxidation rate, expressed in grams per minute (g/min), will be calculated using the Frayn stoichiometric equation from oxygen consumption (VO2) and carbon dioxide production (VCO2) measured by indirect calorimetry during 30 minutes of cycling at 65% of the participant's individual VO2max. Fat oxidation will be compared between the high and low aerobic fitness groups under fasted conditions and between the fasted and fed conditions within the high aerobic fitness group.	During each 30-minute submaximal cycling test performed at 65% of VO2max.
Carbohydrate Oxidation Rate During Submaximal Exercise	Whole-body carbohydrate oxidation rate, expressed in grams per minute (g/min), will be calculated using the Frayn stoichiometric equation from oxygen consumption (VO2) and carbon dioxide production (VCO2) measured by indirect calorimetry during 30 minutes of cycling at 65% of the participant's individual VO2max. Carbohydrate oxidation will be compared between the high and low aerobic fitness groups under fasted conditions and between the fasted and fed conditions within the high aerobic fitness group.	During each 30-minute submaximal cycling test performed at 65% of VO2max.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Resting Fat Oxidation Rate	Resting fat oxidation rate, expressed in grams per minute (g/min), will be calculated from oxygen consumption (VO2) and carbon dioxide production (VCO2) measured by indirect calorimetry during the resting metabolic assessment. Resting fat oxidation will be compared between the high and low aerobic fitness groups under fasted conditions and between the fasted and fed conditions within the high aerobic fitness group.	During the resting metabolic assessment before the fasted and fed exercise conditions.
Resting Carbohydrate Oxidation Rate	Resting carbohydrate oxidation rate, expressed in grams per minute (g/min), will be calculated from oxygen consumption (VO2) and carbon dioxide production (VCO2) measured by indirect calorimetry during the resting metabolic assessment. Resting carbohydrate oxidation will be compared between the high and low aerobic fitness groups under fasted conditions and between the fasted and fed conditions within the high aerobic fitness group.	During the resting metabolic assessment before the fasted and fed exercise conditions.
PBMC Glycolytic Proton Efflux Rate (glycoPER)	Glycolytic proton efflux rate (glycoPER) will be calculated from the total proton efflux rate after correction for the mitochondrial contribution to extracellular acidification. Measurements will be obtained from PBMCs isolated from venous blood samples collected immediately before and immediately after the fasted submaximal exercise test using a Seahorse XFe96 extracellular flux analyzer.	Immediately before and immediately after the fasted 30-minute cycling test at 65% of VO2max.
Resting Metabolic Rate	Resting metabolic rate, expressed in kilocalories per day (kcal/day), will be measured using a Q-NRG indirect calorimetry system in canopy mode. Measurements will be performed after an overnight fast in both aerobic fitness groups. In the high aerobic fitness group, resting metabolic rate will also be reassessed one hour after the ad libitum meal and before the fed exercise test.	Before fasted exercise in both groups and 1 hour after the meal before fed exercise in the high-fitness group.

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Rating of Perceived Exertion During Submaximal Exercise	Perceived exertion will be rated using the Borg 6-20 scale during each 30-minute submaximal cycling test. Higher scores indicate a greater perceived level of exercise intensity and effort.	Every 5 minutes during each 30-minute submaximal cycling test.
Ad Libitum Energy Intake	Total energy intake will be determined from the amount of the standardized pasta-and-meatball meal consumed ad libitum by participants in the high aerobic fitness group. The meal will be prepared using standardized recipes and ingredient quantities to allow quantification of the energy consumed.	During the ad libitum meal provided 1 hour after completion of the fasted exercise test.

Collaborators and Investigators

• Sponsor: Hacettepe University
• Investigators: Principal Investigator: Süleyman BULUT, PhD, Hacettepe University, Faculty of Sport Sciences; Principal Investigator: Muhammed M ATAKAN, PhD, Hacettepe University, Faculty of Sport Sciences

Publications and helpful links

General Publications

• Atakan MM, Guzel Y, Bulut S, Kosar SN, McConell GK, Turnagol HH. Six high-intensity interval training sessions over 5 days increases maximal oxygen uptake, endurance capacity, and sub-maximal exercise fat oxidation as much as 6 high-intensity interval training sessions over 2 weeks. J Sport Health Sci. 2021 Jul;10(4):478-487. doi: 10.1016/j.jshs.2020.06.008. Epub 2020 Jun 18.
• Hedges CP, Woodhead JST, Wang HW, Mitchell CJ, Cameron-Smith D, Hickey AJR, Merry TL. Peripheral blood mononuclear cells do not reflect skeletal muscle mitochondrial function or adaptation to high-intensity interval training in healthy young men. J Appl Physiol (1985). 2019 Feb 1;126(2):454-461. doi: 10.1152/japplphysiol.00777.2018. Epub 2018 Dec 20.
• Mookerjee SA, Goncalves RLS, Gerencser AA, Nicholls DG, Brand MD. The contributions of respiration and glycolysis to extracellular acid production. Biochim Biophys Acta. 2015 Feb;1847(2):171-181. doi: 10.1016/j.bbabio.2014.10.005. Epub 2014 Oct 27.
• Theall B, Stampley J, Cho E, Granger J, Johannsen NM, Irving BA, Spielmann G. Impact of acute exercise on peripheral blood mononuclear cells nutrient sensing and mitochondrial oxidative capacity in healthy young adults. Physiol Rep. 2021 Dec;9(23):e15147. doi: 10.14814/phy2.15147.
• Liepinsh E, Makarova E, Plakane L, Konrade I, Liepins K, Videja M, Sevostjanovs E, Grinberga S, Makrecka-Kuka M, Dambrova M. Low-intensity exercise stimulates bioenergetics and increases fat oxidation in mitochondria of blood mononuclear cells from sedentary adults. Physiol Rep. 2020 Jun;8(12):e14489. doi: 10.14814/phy2.14489.
• Janssen JJE, Lagerwaard B, Porbahaie M, Nieuwenhuizen AG, Savelkoul HFJ, van Neerven RJJ, Keijer J, de Boer VCJ. Extracellular flux analyses reveal differences in mitochondrial PBMC metabolism between high-fit and low-fit females. Am J Physiol Endocrinol Metab. 2022 Feb 1;322(2):E141-E153. doi: 10.1152/ajpendo.00365.2021. Epub 2022 Jan 10.

Study record dates

Study Major Dates

• Study Start (Estimated): November 15, 2026
• Primary Completion (Estimated): November 15, 2027
• Study Completion (Estimated): December 15, 2027

Study Registration Dates

• First Submitted: September 4, 2026
• First Submitted That Met QC Criteria: September 4, 2026
• First Posted (Actual): September 10, 2026

Study Record Updates

• Last Update Posted (Actual): September 10, 2026
• Last Update Submitted That Met QC Criteria: September 4, 2026
• Last Verified: September 1, 2026

More Information

Terms related to this study

• Keywords: Aerobic Fitness; Substrate Oxidation; Fat Oxidation; Carbohydrate Oxidation; Peripheral Blood Mononuclear Cells; Acute Exercise; Indirect Calorimetry; Metabolic Flexibility; Maximal Oxygen Uptake; Mitochondrial Respiration; Metabolic Fuel Utilization; Fasted Exercise
• Other Study ID Numbers: SBA 26/387

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO
• IPD Plan Description: Individual participant data will not be shared due to participant confidentiality and the absence of institutional data sharing infrastructure.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No