This randomized controlled trial will investigate the effects of a 6-week polarized aerobic training intervention on vascular and cardiorespiratory function in apparently healthy sedentary adults aged 30-50 years. Polarized training is characterized by a high proportion of low-intensity aerobic exercise combined with a smaller volume of high-intensity interval exercise, potentially providing complementary haemodynamic and physiological stimuli that support vascular adaptations and cardiovascular health. Although polarized training has been extensively studied in athletic populations, its effects on vascular function in sedentary adults remain insufficiently understood.

Thirty participants will be randomly assigned in a 1:1 ratio to either a supervised polarized aerobic training group or a non-exercising control group. The intervention will consist of a 6-week supervised cycle ergometer training program performed three times per week and prescribed according to individual ventilatory thresholds obtained during cardiopulmonary exercise testing. Training sessions will combine low-to-moderate-intensity continuous exercise and high-intensity interval exercise. Participants allocated to the control group will maintain their habitual sedentary lifestyle throughout the study period.

The primary outcome will be endothelial function assessed by brachial artery flow-mediated dilation. Secondary outcomes will include arterial stiffness assessed by pulse wave velocity, central haemodynamics, microvascular function, cardiorespiratory fitness, skeletal muscle function, pulmonary function, respiratory muscle strength, and circulating biomarkers related to vascular health. Outcome measures will be assessed at baseline and after completion of the 6-week intervention period. The findings of this study may improve understanding of vascular and systemic adaptations to polarized aerobic training and help inform future exercise-based strategies for cardiovascular risk reduction in apparently healthy sedentary adults.

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 knee extensors. Each contraction will be sustained for approximately 3 seconds, with a 1-minute rest interval between attempts. The highest recorded torque value will be used for analysis. Throughout testing, participants will receive continuous verbal encouragement and real-time visual feedback to facilitate maximal effort.

Blood Markers Venous blood samples will be collected by trained healthcare professionals under standardised conditions. Sampling will be performed at two time points: at baseline (week 0) and following the intervention (week 6). All measurements will be conducted under consistent laboratory conditions in accordance with established recommendations for biological sampling.

Circulating biomarkers will be analysed to reflect key physiological pathways related to vascular function, including endothelial function, inflammation, and oxidative stress, and cardiometabolic regulation (e.g., nitric oxide-related indices, inflammatory markers), in line with the mechanistic framework of the study.

For biochemical analyses, venous blood samples will be collected at both time points. For molecular analyses, blood will be collected in EDTA tubes, and genomic DNA will be isolated from whole blood using standardised procedures. The isolated DNA will be used to assess selected genetic variants (single-nucleotide polymorphisms, SNPs) in candidate genes using quantitative polymerase chain reaction (qPCR)-based methods. Candidate genes will be selected based on current scientific evidence and relevant biological databases, and genotype determination will be performed using allele discrimination analysis with appropriate reference controls.

Intervention and control Participants allocated to the intervention group will undergo a 6-week supervised polarized (POL) aerobic training programme performed on a cycle ergometer (Concept2 BikeErg; Concept2). The intervention will consist of three weekly exercise sessions performed on non-consecutive days and will combine low-to-moderate-intensity continuous training (LMICT) with high-intensity interval training (HIIT). Exercise intensity will be individually prescribed according to ventilatory thresholds determined during baseline cardiopulmonary exercise testing. Specifically, continuous aerobic exercise intensity will be prescribed relative to the gas exchange threshold (GET), whereas high-intensity intervals will be prescribed relative to the respiratory compensation point (RCP). The weekly training structure will include two HIIT sessions and one continuous aerobic training session, thereby creating a polarized intensity distribution characterized by a high proportion of low-intensity exercise, a smaller volume of high-intensity exercise, and minimal exposure to the moderate-intensity domain.

The LMICT session will include a 5-minute warm-up at 1 W/kg, followed by 30 minutes of continuous cycling at 90% to 110% of the heart rate corresponding to GET. HIIT sessions will include a 5-minute warm-up at 1 W/kg, followed by four 4-minute intervals performed at 100% to 105% of the heart rate corresponding to RCP, interspersed with 3 minutes of active recovery at 1 W/kg. Cadence will be maintained between 60 and 90 rpm across all sessions. In addition to individualized heart rate targets, participants will be provided with corresponding power output targets expressed in W at the beginning of each training session. To account for cardiovascular drift during exercise, training intensity will be maintained within predefined heart rate ranges to ensure an adequate physiological stimulus while preserving individualisation. Internal training load will be quantified using session rating of perceived exertion (RPE) using a 10-point scale to support monitoring of intervention fidelity. All exercise sessions will be supervised by trained personnel holding at least a master's degree in kinesiology or a related field to ensure adherence to the intervention protocol. This threshold-based training approach is consistent with current evidence-based exercise prescription recommendations.

Based on the planned weekly structure, the overall training intensity distribution is expected to approximate a polarized profile, with approximately 75% to 80% of total training time performed within the low-intensity domain, 15% to 20% at high intensity, and minimal exposure (<10%) to the moderate-intensity threshold domain.

Participants allocated to the control group will be instructed to maintain their habitual sedentary behaviour throughout the 6-week intervention period and to refrain from initiating any structured exercise training. To monitor compliance, habitual physical activity patterns will be assessed using a wearable device (Polar Vantage V3; Polar Electro, Kempele, Finland), including continuous monitoring of daily step count and heart rate responses. Participants requiring medical treatment during the study will not be excluded; however, all concomitant therapies and medical changes occurring during the intervention period will be documented and considered during statistical analyses.

Inclusion Criteria:

• Men and women aged 30-50 years
• Apparently healthy individuals without diagnosed cardiovascular, metabolic, or renal disease
• Sedentary lifestyle verified using the Global Physical Activity Questionnaire (GPAQ) and current physical activity classification criteria
• Ambulatory blood pressure values within the normotensive range (<140 mmHg systolic and <90 mmHg diastolic)
• Body mass index (BMI) <30 kg/m²
• No regular participation in structured exercise training

Exclusion Criteria:

• Ambulatory blood pressure values ≥140 mmHg systolic and/or ≥90 mmHg diastolic
• Moderate or high physical activity level according to the Global Physical Activity Questionnaire (GPAQ)
• Diagnosed chronic cardiovascular, metabolic, renal, respiratory, or other systemic disease
• Use of prescribed medication influencing cardiovascular or metabolic function
• Musculoskeletal injury of the lower extremities within the previous six months
• Menopause
• Absence of a regular menstrual cycle
• Pregnancy
• Current smoking or tobacco use

De-identified individual participant data related to the reported study outcomes will be made available following publication of the primary study results. The study protocol will be made publicly available prior to publication.

The data and supporting materials will become available following publication of the primary study results and are expected to remain accessible through the institutional repository of the University of Ljubljana.

De-identified data and study protocol will be accessible to researchers and the broader scientific community through the institutional repository of the University of Ljubljana. Data will be available for scientifically valid research purposes without restrictions.

Outcome assessors will be blinded to group allocation whenever possible.