Polarized Aerobic Training and Vascular Adaptations in Apparently Healthy Individuals

This study is a work package of the ACT-ON project and is designed to evaluate the effects of a 6-week polarized aerobic training intervention on vascular and cardiorespiratory function in apparently healthy sedentary adults. In addition, the study will investigate physiological adaptations across multiple cardiovascular, microvascular, neuromuscular, respiratory, and biochemical domains using a comprehensive multimodal assessment approach. The trial will be conducted as a single-centre, assessor-blinded, parallel-group randomized controlled trial comprising an intervention group and a non-exercising control group.

Thirty apparently healthy sedentary men and women aged 20-50 years will be recruited and randomly assigned in a 1:1 ratio to either a polarized aerobic training intervention or a non-exercising control condition. Eligible participants must not have diagnosed cardiovascular, metabolic, or renal disease, must not use medications influencing cardiovascular function, and must not engage in regular exercise training. Sedentary status will be verified using the Global Physical Activity Questionnaire (GPAQ) and current physical activity classification criteria. Participants with ambulatory blood pressure values exceeding 139 mmHg systolic and/or 89 mmHg diastolic, body mass index ≥30 kg/m², or other contraindications to exercise testing will be excluded.

Prior to enrollment, all interested individuals will attend a screening visit to confirm eligibility and ensure safe participation. The visit will be conducted in the morning and will include measurements of resting blood pressure (M7, Omron, Japan), body height (GPM, Model 101, Switzerland), body composition (InBody 720, Biospace, South Korea), and completion of the GPAQ. Female participants will additionally complete a Reproductive Status Questionnaire (RSQ) to further assess eligibility. Participants will be instructed to avoid vigorous physical activity, alcohol, and caffeine in the days preceding the visit and to arrive well rested and in a fasted state. Following the screening assessments, participants will be fitted with a 24-hour ambulatory blood pressure monitor (Boso TM-2430) to characterize habitual blood pressure status and minimize the potential influence of the white-coat effect. Written informed consent will be obtained from all participants prior to any study-related procedures.

Following enrollment and randomization, all participants will undergo comprehensive physiological assessments at the Faculty of Sport, University of Ljubljana. Baseline testing will encompass vascular and haemodynamic function, microvascular reactivity, cardiorespiratory fitness assessed by cardiopulmonary exercise testing for determination of ventilatory thresholds, skeletal muscle function, pulmonary function, respiratory muscle strength, resting blood pressure, and circulating biomarkers related to cardiovascular health and physiological adaptation to exercise.

All assessments will be performed under standardized laboratory conditions by trained investigators blinded to group allocation. Measurements of vascular and haemodynamic function will be conducted in a quiet, temperature-controlled environment (22-25 °C). Prior to testing, participants will rest in the supine position for at least 15 minutes to ensure haemodynamic stabilisation and will be instructed to refrain from caffeine, alcohol, and strenuous physical activity for at least 48 hours before each testing session. The same procedures will be repeated at least 72 hours after the final exercise session upon completion of the 6-week intervention period.

Participants allocated to the intervention group will undertake a 6-week supervised polarized aerobic training program performed on a cycle ergometer three times per week. The program will combine low-to-moderate-intensity continuous exercise and high-intensity interval training prescribed according to individual ventilatory thresholds. Participants allocated to the control group will maintain their habitual sedentary lifestyle throughout the intervention period and will refrain from initiating structured exercise training.

Primary outcomes Endothelial function Endothelial function will be assessed concurrently using flow-mediated dilation (FMD) and flow-mediated slowing (FMS), representing complementary measures of conduit artery vasodilation and dynamic changes in arterial stiffness.

Brachial artery FMD will be assessed using high-resolution ultrasound imaging (Acuson Juniper; Siemens, Germany) with a linear-array transducer (7-12 MHz), in accordance with current expert consensus guidelines. The brachial artery will be imaged longitudinally 1-3 cm proximal to the antecubital fossa, with the left arm positioned in a supported, slightly abducted (70-80°) position to minimise movement. After a 3-minute baseline recording period, a pneumatic cuff placed on the forearm or upper arm will be inflated to 50 mmHg above the resting systolic blood pressure for 5 minutes to induce ischemia. Upon cuff release, continuous ultrasound recording (4 minutes) will capture the reactive hyperaemic response, and the peak arterial diameter will be determined. FMD will be expressed as the relative (%) increase in peak arterial diameter following cuff release compared with baseline diameter.

Simultaneously, FMS will be assessed using an oscillometric device (Vicorder, 80 Beats Medical, Berlin, Germany), enabling continuous measurement of pulse wave velocity (PWV) during the occlusion-reperfusion sequence. Cuffs will be positioned according to manufacturer guidelines for brachial-radial PWV assessment. During both the baseline and post-occlusion phases, the brachial and radial cuffs will be inflated to a sub-occlusive pressure (\<65 mmHg) to allow simultaneous recording of brachial and radial pulse waveforms. Brachial-radial PWV will be calculated every 3-4 seconds during the baseline and post-occlusion periods. Mean baseline PWV will be calculated as the average PWV during the baseline period, while mean PWV values will be computed for each 30-second interval during the post-occlusion phase. FMS will be expressed as the relative (%) reduction in PWV following cuff release compared with baseline PWV values.

Secondary outcomes Arterial stiffness and pulse wave analysis Arterial stiffness and central haemodynamics will be assessed using an oscillometric system (Vicorder, software version 4; Skidmore Medical, United Kingdom). Pulse wave analysis will be performed at the brachial artery using a pneumatic cuff inflated to a sub-diastolic pressure of 65 mmHg, enabling non-invasive acquisition of arterial pressure waveforms via a volume displacement method. Central aortic pressure indices, including aortic pulse pressure and mean arterial pressure, will be derived using a validated brachial-to-aortic transfer function incorporated within the device software. The augmentation index (AIx) will be calculated from the reconstructed central pressure waveform as an indicator of wave reflection. To enhance measurement reliability, multiple consecutive waveforms (10 consecutive beats) will be recorded and averaged.

Arterial stiffness will be further assessed using pulse wave velocity (PWV) across three arterial segments: brachial-radial (brPWV), femoral-ankle (faPWV), and carotid-femoral (cfPWV). Measurements will be obtained using standardised cuff placements and simultaneous waveform acquisition. For each segment, the arterial path length will be measured according to anatomical landmarks and manufacturer recommendations, and PWV will be calculated as the ratio of distance to pulse transit time.

Microvascular function Microvascular function will be assessed using near-infrared spectroscopy (NIRS) during the vascular occlusion-reperfusion protocol. A continuous-wave NIRS system (PortaMon; Artinis Medical Systems, The Netherlands) will be used to quantify relative changes in oxygenated (O₂Hb) and deoxygenated haemoglobin (HHb). The NIRS sensor will be positioned over the forearm musculature (flexor region) and secured to minimise movement artefacts. Signals will be recorded continuously throughout baseline, occlusion, and reperfusion phases. The tissue saturation index (TSI), reflecting the balance between oxygen delivery and utilisation, will be derived from the NIRS signal. Microvascular reactivity will be evaluated based on the reoxygenation response following cuff release. The average values of the first 10 seconds following reperfusion will be used to calculate microvascular reactivity, consistent with established literature.

Cardiorespiratory fitness Cardiorespiratory fitness will be assessed using a graded cardiopulmonary exercise test performed on an electronically braked cycle ergometer (Excalibur Sport; Lode, The Netherlands). Following a 5-minute standardised warm-up at an intensity of 1 W/kg, participants will complete a ramp incremental protocol starting at 60 W, with workload increasing by 15 W/min until volitional exhaustion. Participants will be instructed to maintain a stable self-selected cadence between 70 and 95 revolutions per minute throughout the test. Respiratory gas exchange variables, including oxygen uptake (VO₂), carbon dioxide production (VCO₂), and minute ventilation (VE), will be measured continuously using a breath-by-breath metabolic cart (Quark CPET, Cosmed, Italy), while heart rate will be recorded simultaneously using a chest-strap monitor (Polar H10; Polar Electro, Finland). Standardised verbal encouragement will be provided to ensure maximal effort.

Maximal oxygen uptake (VO2max) will be defined as the highest value achieved during the test based on established criteria. Gas exchange threshold (GET) and respiratory compensation point (RCP), along with the corresponding heart rate and power output values, will be determined from gas exchange data in accordance with current methodological recommendations and used for exercise prescription and subsequent analyses.

Skeletal muscle mechanical properties Skeletal muscle mechanical properties will be assessed non-invasively using a handheld myotonometer (MyotonPRO; Myoton AS, Estonia), which enables quantification of biomechanical and viscoelastic characteristics of muscle tissue. Measurements will be performed with participants in a relaxed, standardised position to ensure that the assessed muscles of the quadriceps femoris remain in a passive state. The probe will be positioned perpendicular to the skin surface, and brief mechanical impulses will be applied to induce natural oscillations within the tissue. Parameters reflecting muscle tone, stiffness, and viscoelastic properties will be derived for analysis.

Pulmonary function and respiratory muscle strength Pulmonary function will be assessed using spirometry (Alpha Spirometer; Vitalograph, UK) in accordance with established guidelines. Participants will be tested in a seated position while wearing a nose clip. Standard spirometric indices, including forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), vital capacity (VC), and peak expiratory flow (PEF), will be obtained from repeated trials, with the highest acceptable value retained for analysis.

Respiratory muscle strength will be assessed using maximal inspiratory (MIP) and maximal expiratory (MEP) pressure measurements. Multiple trials will be performed under standardised conditions, with adequate rest between efforts (1 minute), and the highest valid value will be used for analysis.

Maximal isometric muscle strength Maximal voluntary isometric contraction (MVIC) of the knee extensors will be assessed to evaluate changes in muscle strength over time. Torque will be measured using an isometric dynamometry system (CP Trainer; CompletePerformance, Serbia). Participants will first perform two submaximal familiarisation contractions to ensure proper understanding of the procedure, followed by two maximal isometric contractions of the

• Aortic pulse pressure (AOPP) [Срок оценки: Baseline and post-intervention (week 6; at least 72 hours after the final exercise session)]
• Augmentation index [Срок оценки: Baseline and post-intervention (week 6; at least 72 hours after the final exercise session)]
• Muscle tissue reoxygenation slope (microvascular reactivity) [Срок оценки: Baseline and post-intervention (week 6; at least 72 hours after the final exercise session)]
• Skeletal muscle stiffness [Срок оценки: Baseline and post-intervention (week 6; at least 72 hours after the final exercise session)]
• Forced vital capacity (FVC) [Срок оценки: Baseline and post-intervention (week 6; at least 72 hours after the final exercise session)]
• Forced expiratory volume in one second (FEV1) [Срок оценки: Baseline and post-intervention week 6; at least 72 hours after the final exercise session.]
• Vital capacity (VC) [Срок оценки: Baseline and post-intervention week 6; at least 72 hours after the final exercise session.]
• Peak expiratory flow (PEF) [Срок оценки: Baseline and post-intervention week 6; at least 72 hours after the final exercise session.]
• Maximal inspiratory pressure (MIP) [Срок оценки: Baseline and post-intervention (week 6; at least 72 hours after the final exercise session)]
• Maximal expiratory pressure (MEP) [Срок оценки: Baseline and post-intervention week 6; at least 72 hours after the final exercise session.]
• Blood biomarkers [Срок оценки: Baseline and post-intervention (week 6; at least 72 hours after the final exercise session)]
• Blood pressure [Срок оценки: Resting blood pressure: baseline and post-intervention (week 6; at least 72 hours after the final exercise session); 24-hour ambulatory blood pressure monitoring: baseline only]