Effects of Food Intake Sequence on Substrate Utilisation and Endurance Performance

Effects of Manipulating Food Intake Sequence in the Pre-exercise Meal on Substrate Utilisation and Endurance Performance in Athletes

The goal of this clinical trial is to understand how consuming a pre-exercise meal in different food intake sequences affects substrate utilisation and endurance exercise performance in healthy trained athletes. Specifically, the main questions it aims to answer are:

• Does consuming the pre-exercise meal in different food intake sequences alter the whole-body utilisation of fat and CHO at rest and during prolonged exercise at moderate to heavy intensities?
• Does consuming the pre-execise meal in different food intake sequences alter time-trial performance?
• Does consuming the pre-exercise meal in different food intake sequences alter other metabolic and physiological responses before, during or after exercise (i.e., blood glucose, blood lactate, heart rate (HR), ratings of perceived exertion (RPE), appetite)?

To address these questions, researchers will compare eating the dietary sources of rapidly absorbed CHO at the end (CHO-last meal pattern) or at the start (CHO-first meal pattern) of a standardised pre-exercise meal, in a randomised, counterbalanced, crossover design. Two hours after starting the pre-exercise meal, participants will perform a 60-min submaximal test on a cycling ergometer at two different intensities (30 min at 90% of the ventilatory threshold (VT) 1 and 30 min at 50% VT1-VT2), followed by a 10-km cycling time trial (TT).

In the screening visit participants will:

• Be screened for cardiometabolic conditions
• Perform an 8-12-min graded exercise test to determine their maximal pulmonary oxygen uptake (VO2max) and non-invasive submaximal anchors of performance (VT 1 and VT 2)
• Be familiarised to the experimental procedures

In experimental visits participants will:

• Follow guidelines pertaining to dietary intake, exercise and sleep in the previous 24 hours
• Have a standardised test meal in either the CHO-last or CHO-first food intake sequence
• Have 3 drops of capillary blood (0,9 µL in total) collected to measure glucose and lactate pre-meal, 15, 30, 45, 60, 90 and 120 min post-meal, every 15 min during submaximal exercise and post-TT
• Breathe into an indirect calorimetry mask for 5 min pre-meal, 15, 30, 45, 60, 90 and 120 min post-meal, every 15 min during submaximal exercise and post-TT
• Wear a HR monitor continuously during submaximal exercise and the TT
• Rate their appetite on visual analogue scales pre-meal, 15, 30, 45, 60, 90 and 120 min post-meal, and 30 min post-TT
• Rate their perceived exertion on RPE scales every 15 min during submaximal exercise, post-TT and 30 min post-TT.

Data will be compared within-participant between food intake sequences using linear mixed-effects models with random intercepts, to account for repeated measurements, interindividual variability, and potential missing data.

Study Overview

• Status: Recruiting
• Conditions: Healthy Participants; Endurance Exercise; Substrate Metabolism
• Intervention / Treatment: Other: Carbohydrate-last meal pattern; Other: Carbohydrate-first meal pattern

• Study Type: Interventional
• Enrollment (Estimated): 19
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: João Brito, PhD; Email: joao.brito@fpf.pt
• Study Contact Backup: Name: Rita Giro; Phone Number: 351 225074320; Email: up201307940@edu.fcna.up.pt

Study Locations

• Portugal
  • Oeiras
    • Lisbon, Oeiras, Portugal, 1495-433
      • Recruiting
      • Cidade do Futebol
      • Contact: João Brito, PhD; Phone Number: +351 213252700; Email: joao.brito@fpf.pt

Participation Criteria

Eligibility Criteria

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

Description

Inclusion Criteria:

• Adult (18-64 years old)
• Men
• Endurance-trained athletes (meeting training and performance caliber criteria ≥Tier 2; McKay et al., 2022)
• Healthy (meeting the exclusion criteria for medical conditions)
• Normal glucose tolerant according to the latest criteria established by the American Diabetes Association (2024): HbA1c <5.7%, fasting plasma glucose <5.6 mmol/L (100 mg/dL) or 2-h plasma glucose <7.8 mmol/L (140 mg/dL) during a 75-g OGTT
• Able and willing to provide informed consent and safely comply with study procedures

Exclusion Criteria:

• Any medical condition or behaviour deemed either to pose undue personal risk to the participant or introduce bias into the experiment (e.g. cardiovascular disease; alcohol or substance abuse; any condition affecting the glucose or lipid metabolism, reviewed on a case by case basis)
• Any reported medication or supplementation that may interfere with the glucose metabolism (e.g., acarbose, insulin, metformin, semaglutide, thiazolidinediones, sulfonylureas, corticosteroids, thiazide diuretics) or lipid metabolism (e.g., statins, nicotinic acid, colestyramine anhydrous, ezetimibe, fibrates, L-carnitine). Other medication and supplementation will be reviewed on a case by case basis.
• Known food allergy, intolerance or hypersensitivity to any of the test meal ingredients
• Recent change in body mass (± 2 kg in the last 2 months)
• Smoking

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Randomized
• Interventional Model: Crossover Assignment
• Masking: Single

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: CL-CF; The interventions (carbohydrate-last meal pattern (CL) or carbohydrate-first meal pattern (CF)) will be delivered in random order at two study visits, separated by a wash-out period of at least 7 days. If participants are allocated to this study arm, they will receive the CL on the first visit and the CF on the second visit. The nutritional composition of the test-meal will comply to the current sports nutrition guidelines for the pre-exercise meal (i.e., 1-4 g of CHO/kg of body mass, moderate amounts of protein, low-fat and fibre content, 5-10 mL/kg of body mass of fluids) (Thomas et al., 2016). Participants will be encouraged to consume the meal in full within 15 minutes, at a comfortable pace, to mimic real eating circumstances.	Other: Carbohydrate-last meal pattern; Skyr yoghurt, whey protein and almonds over ~5 min, immediately followed by white bread, strawberry jam, banana and pulp-free orange juice over ~10 min.; Other Names: CL; CHO-last; Other: Carbohydrate-first meal pattern; White bread, strawberry jam, banana and pulp-free orange juice over ~10 min, immediately followed by skyr yoghurt, whey protein and almonds over ~5 min.; Other Names: CF; CHO-first
Active Comparator: CF-CL; The interventions (carbohydrate-last meal pattern (CL) or carbohydrate-first meal pattern (CF)) will be delivered in random order at two study visits, separated by a wash-out period of at least 7 days. If participants are allocated to this study arm, they will receive the CF on the first visit and the CL on the second visit. The test-meal will be identical and isocaloric in both interventions of the study.	Other: Carbohydrate-last meal pattern; Skyr yoghurt, whey protein and almonds over ~5 min, immediately followed by white bread, strawberry jam, banana and pulp-free orange juice over ~10 min.; Other Names: CL; CHO-last; Other: Carbohydrate-first meal pattern; White bread, strawberry jam, banana and pulp-free orange juice over ~10 min, immediately followed by skyr yoghurt, whey protein and almonds over ~5 min.; Other Names: CF; CHO-first

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Whole-body fat oxidation	Difference between CHO-last and CHO-first meal patterns in the whole-body fat oxidation rate (g/min) over time adjusted for baseline, measured by indirect calorimetry.	Every 15 minutes post-meal (0-120 minutes), every 15 minutes throughout the subsequent standardised exercise protocol (120-180 minutes)
Time-trial performance	Difference between CHO-last and CHO-first meal patterns in the time to complete (min:sec) a 10-km cycling TT, assessed using a validated cycle ergometer.	Immediately upon completion of the time trial subsequent to the post-meal resting period (0-120 minutes) and standardised exercise protocol (120-180 minutes)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Time-trial power output	Difference between CHO-last and CHO-first meal patterns in the average power output (W) during a 10-km cycling TT, assessed using a validated cycle ergometer.	Immediately upon completion of the time trial subsequent to the post-meal resting period (0-120 minutes) and standardised exercise protocol (120-180 minutes)
Whole-body CHO oxidation	Difference between CHO-last and CHO-first meal patterns in the whole-body CHO oxidation rate (g/min) over time adjusted for baseline, measured by indirect calorimetry.	Every 15 minutes post-meal (0-120 minutes), every 15 minutes throughout the subsequent standardised exercise protocol (120-180 minutes)
Respiratory exchange ratio (RER)	Difference between CHO-last and CHO-first meal patterns in the RER over time adjusted for baseline, measured by indirect calorimetry.	Every 15 minutes post-meal (0-120 minutes), every 15 minutes throughout the subsequent standardised exercise protocol (120-180 minutes)
Energy expenditure	Difference between CHO-last and CHO-first meal patterns in the energy expenditure rate (kcal/min) over time adjusted for baseline, measured by indirect calorimetry.	Every 15 minutes post-meal (0-120 minutes), every 15 minutes throughout the subsequent standardised exercise protocol (120-180 minutes)
Blood glucose concentrations	Difference between CHO-last and CHO-first meal patterns in the capillary blood glucose concentrations (mmol/L) over time adjusted for baseline, measured by a validated glucometer.	Every 15 minutes post-meal in the first hour (0-60 min), every 30 minutes in the second hour (60-120 min), every 15 minutes throughout the standardised exercise protocol (120-180 min) and immediately upon completion of the subsequent time trial
Blood lactate concentrations	Difference between CHO-last and CHO-first meal patterns in the capillary blood lactate concentrations (mmol/L) over time adjusted for baseline, measured by a validated lactate meter.	Every 15 minutes post-meal in the first hour (0-60 min), every 30 minutes in the second hour (60-120 min), every 15 minutes throughout the standardised exercise protocol (120-180 min) and immediately upon completion of the subsequent time trial
Heart Rate	Difference between CHO-last and CHO-first meal patterns in the heart rate (bpm) over time adjusted for baseline, assessed as 15-minute averages during exercise using a validated heart rate monitor.	Continuously throughout the standardised exercise protocol (120-180 minutes) and subsequent time trial
Ratings of perceived exertion (RPE)	Difference between CHO-last and CHO-first meal patterns in the absolute RPE over time, assessed during and after exercise using the validated modified Borg's 0-10 scale.	Every 15 minutes during the standardised exercise protocol (120-180 minutes), immediately upon completion of the subsequent time trial, 30 minutes after completion of the time trial
Subjective appetite ratings	Difference between CHO-last and CHO-first meal patterns in the appetite ratings (mm) over time adjusted for baseline, assessed at rest and after exercise using validated 100-mm visual analogue scales.	Every 15 minutes post-meal in the first hour (0-60 minutes), every 30 minutes in the second hour (60-120 minutes), and 30 minutes after completion of the time trial

Collaborators and Investigators

• Sponsor: Universidade do Porto
• Collaborators: Fundação para a Ciência e a Tecnologia; Federação Portuguesa de Futebol
• Investigators: Principal Investigator: Rita Giro, FCNAUP, University of Porto; Federação Portuguesa de Futebol

Study record dates

Study Major Dates

• Study Start (Actual): April 1, 2026
• Primary Completion (Estimated): July 1, 2026
• Study Completion (Estimated): July 1, 2026

Study Registration Dates

• First Submitted: November 14, 2025
• First Submitted That Met QC Criteria: March 26, 2026
• First Posted (Actual): March 31, 2026

Study Record Updates

• Last Update Posted (Actual): April 3, 2026
• Last Update Submitted That Met QC Criteria: April 2, 2026
• Last Verified: April 1, 2026

More Information

Terms related to this study

• Keywords: glucose metabolism; food order; meal sequence; sport nutrition; substrate utilisation
• Other Study ID Numbers: FOODSEQ-SUEP; 2022.12954.BD (Other Grant/Funding Number: Fundação para a Ciência e Tecnologia)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: Anonymised IPD will be made available upon reasonable request directed to the corresponding author.
• IPD Sharing Time Frame: IPD will become accessible following the publication of all study findings.
• IPD Sharing Access Criteria: IPD will be made available to researchers with legitimate scientific purposes (such as systematic reviews or meta-analyses).
• IPD Sharing Supporting Information Type: SAP; ICF

Drug and device information, study documents

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