This study is a work package of the ACT-ON project, aimed at establishing the reliability and validity of flow-mediated slowing and microvascular function, as well as determine the acute response of different vascular function measures to different exercise conditions. The investigators aimed to include vascular function measures evaluating different parts of the vasculature (e.g. central, peripheral and microvasculature) to establish whether the specific response differs between healthy participants and participants at risk of developing cardiovascular diseases.
Data collection will be conducted using a range of instruments and tools in the following order.
To ensure compliance with the inclusion criteria, participants will undergo a screening visit prior to the enrollment in the study. The visit will take place in the morning including the following assessments: Standardized blood pressure (M6, Omron, Japan), height (GPM, Model 101, Switzerland) and weight (InBody 720, Biospace, South Korea) measurement, as well as the administration of the GPAQ questionnaire. Female participants will additionally complete a Reproductive status Questionnaire (RSQ) (Schmalenberger \& Eisenlohr-Moul, 2019) to determine eligibility based on previously established inclusion and exclusion criteria. Participants will be advised to refrain from physical activity, alcohol and caffeine consumption in the days leading up to the visit, as well as to arrive well rested and in a fasted state.
Study protocol
After enrollment in the study, participants will complete three visits with 72h between each visit. The visits will be completed at the same hour of the day to ensure minimal circadian variability. Participants will be advised to refrain from physical exercise and consumption of alcohol and caffeine in the days leading up to the first visit, as well as during the study. Additionally, they will be advised to arrive well rested and to consume a standardized meal 2-3 hours before the visit. To enable physical activity monitoring during the study, participants will wear wrist monitors with 24-h measurement of heart rate on the wrist (Vantage V3, Polar, Finland). In the event that physical activity is detected during the course of the study, participants will be excluded from the study. Additionally, participants will be required to maintain a dietary intake log during the duration of the study.
Body composition measurement using bioimpendance method (InBody 720, Biospace, South Korea) will be taken after arrival to the laboratory of the Institute of Sport, University of Ljubljana. After, participants will sit on a chair for 10 minutes in a quite environment with the hand extended and supinated, resting on a surface at heart level. Subsequently, three consecutive BP measurements will be taken using an automated upper-arm blood pressure monitor (M6, Omron, Japan) with one-minute intervals between each measurement. BP will be calculated as the average of the last two recordings.
Haemodynamics
Pulse Wave Velocity (PWV) Characteristics
The Vicorder device (software version 4; Skidmore Medical, United Kingdom) will be used to measure pulse wave characteristics (PWA) of the brachial artery and pulse wave velocity from the carotid to the femoral artery (cfPWV). This device uses an oscillometric technique to obtain the pulse wave. cfPWV measurements will be taken using a 10 cm cuff around the upper right thigh to detect the femoral pulse and a 3 cm cuff around the neck to detect the right carotid pulse. The neck cuff is designed to be positioned above one carotid artery, avoiding compression of the trachea and both carotid arteries simultaneously. The distance between the suprasternal notch and the cuff on the upper thigh will be measured according to the manufacturer's instructions. Both cuffs will automatically inflate simultaneously to 65 mmHg, with pulse waves recorded in 3 to 5 second intervals while the participant lies in a supine position.
Flow-Mediated Slowing (FMS)
The VICORDER® EndoCheck FMS model will be used for FMS determination. Testing will be conducted following the device instructions. The pulse wave velocity (PWV) on the upper arm was measured simultaneously between the wrist and upper part of the right arm over 10 minutes, with a 5-minute flow occlusion period. FMS will be determined by continuously measuring the minimal PWV compared to the baseline PWV, with the percentage change designated as FMS.
Microvascular function
Near-infrared spectroscopy (NIRS) will be used to assess microvascular function, using a sensor to determine relative changes in oxygenated (O2Hb) and deoxygenated (HHb) haemoglobin. The sensor will be placed on the anteromedial part of the forearm (flexor carpi radialis muscle), 2 cm below the medial epicondyle of the humerus. A continuous-wave NIRS system (PortaMon, Artinis, Netherlands) will be used. The sensor is equipped with six light sources operating at six wavelengths (759, 760, 763, 848, 844 in 848 nm) and an optode distance of 35 mm. The NIRS signal will be recorded at a sampling frequency of 10 Hz. Tissue Saturation Index (TSI) will be calculated based on the modified Beer-Lambert law using the following equation:
TSI= (\[O2Hb\])/((\[HHb\]+\[O2Hb\])) Microvascular function will be assessed concurrently with the FMS measurement. Based on changes of the TSI signal during and after the ischemic stimulus, several variables will be calculated to evaluate microvascular function.
Blood markers
Blood markers will be taken from the left median cubital vein by a licenced clinician following standardized protocol. A 7 mL sample will be taken before the start of vascular function measurements before and after each assigned condition. The analysis of the sample will include basic blood panel markers: total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides, blood glucose.
Physical performance
To obtain physiological parameters, the investigators will use a gas analyzer CPET (COSMED, Italy). The device provides reliable values for oxygen uptake (VO2), carbon dioxide production (VCO2), and pulmonary ventilation (VE) on a breath-by-breath basis. Heart rate will be measured simultaneously with a heart rate monitor chest strap (Polar, model H10, Finland). Data will be recorded in 5-second intervals and automatically analyzed using the original Polar software.
Incremental cycling test
After 5 minutes of baseline measurements sitting on the stationary ergometer (Excalibur sport, Lode, Netherlands) the participants will begin warm-up for 5 minutes at 1 W/kg. After, the test will be performed, in a step-incremental protocol starting at 60 W and increase of 15 W/min every minute.
Exercise Interventions
Participants will undergo the following tests (with 72 hours between visits): a) 30 min of rest (control condition), followed by an incremental exercise test to determine maximal oxygen uptake and exercise intensity prescription based on ventilatory thresholds, and in a randomized fashion: b) An acute moderate-intensity training session, c) an acute high-intensity interval training session. All exercise stimuli will be performed on an electronically braked cycling ergometer. Exercise intensity will be individually adjusted based on exercise test results. The intensity and duration of exercise training sessions will be adjusted to achieve similar caloric expenditure. Participants will be advised to refrain from exercise, consummation of alcohol and caffeine in the days before and during the research period. Additionally, they will be instructed to arrive well rested and consume a standardized meal 2-3 hours before arriving for evaluation. All measurements will be conducted at the same time of day, between 8:00 and 12:00, to minimize circadian variability. Vascular function (PWV, FMS, NIRS, blood markers) will be assessed before the start and after the end of each exercise stimulus and control condition. Blood markers will be collected from the cubital vein before and immediately after exercise.
Participants will undertake 5 min of light-intensity warm-up. Moderate-intensity exercise will be performed as 30 minute of continuous cycling set to 65% of intensity at respiratory compensation point (RCP), which is used as a marker of maximal metabolic steady state during exercise. High-intensity exercise will be performed as 4 repetitions of 4-minute intervals at 95 - 100 % of RCP, with 3 minutes of passive rest. Heart rate will be used as an additional measure to ensure adherence to the correct intensity zone (Coates et al., 2023). During exercise, participants' O2Hb and HHb, as well as TSI response will be monitored using NIRS, along with heart rate and cycling load. To ensure minimal dispersion of the NIRS signal, the adipose tissue thickness (ATT) on the muscle belly of the measured muscles will be assessed and ATT measurements exceeding one half of the optode distance will be excluded. Blood pressure measurements will be taken upon arrival and after each interval, as well as immediately after cessation of exercise. Parameters during exercise will be used to determine the response to different exercise intensities.
