The purpose of this study is to assess the effects of an 8-week supervised high-intensity interval training (HIIT) program (vs. physical activity recommendations according to current guidelines) on a comprehensive panel of circulating sphingolipids in middle-aged females and males at elevated cardiometabolic risk.

Cardiometabolic diseases (CMD) account for about half of all deaths from non-communicable diseases and are responsible for about one-third of all deaths worldwide. To combat the growing burden of CMD on health systems, a shift towards more effective prevention and early detection of these diseases is urgently needed.

Blood lipids have been used since the middle of the last century to determine the risk of developing CMD. Although classically used biomarkers such as cholesterol and triglycerides provide acceptable risk assessment, there is increasing evidence that sphingolipids, particularly ceramides, may allow improved risk assessment. Mechanistically, there is growing data that sphingolipid accumulations lead to atherosclerosis and insulin resistance.

To measure circulating sphingolipids in clinical practice, it is essential to provide patients with evidence-based interventions that reduce sphingolipid levels and quantify the reduction expected from such an intervention. Preliminary data suggest that regular physical activity (PA), an effective, low-cost, and patient-empowering means of health optimisation, may reduce sphingolipid levels.

The current research project aims to explore whether and to what extent a fitness-enhancing high-intensity interval training (HIIT) programme can lower circulating sphingolipid levels in middle-aged individuals at elevated cardiometabolic risk (50% females). An 'omic-scale sphingolipid profiling will be applied to capture the circulating sphingolipidome comprehensively.

Participants will be randomly allocated to either the intervention or the control group. The exercise intervention will consist of an 8-week supervised HIIT (two walking- and one indoor cycling-based session weekly). The control group will get PA recommendations based on current guidelines.

Maximal cardiopulmonary exercise testing (CPET) will be conducted on a cycle ergometer to determine the VO2peak, peak heart rate and peak power output. After the 8-week training programme, a second CPET will be performed to verify if the exercise intervention effectively improved CRF. Body composition will be analysed before and after the 8-week intervention by dual-energy x-ray absorptiometry and by bioelectrical impedance analysis.

Trained medical staff will draw blood samples by venepuncture of the cubital fossa following an overnight fast pre- and post-intervention. Planned blood analyses for basic characterisation of risk factor profiles include total cholesterol, low-density lipoprotein cholesterol (LDL) and high-density lipoprotein cholesterol (LDL), triglycerides, and HbA1c. Glucose and insulin will also be measured to estimate insulin resistance using the HOMA-IR. A high-coverage method using reversed-phase liquid chromatography coupled to tandem mass spectrometry (RPLC-MS/MS) will be applied to quantify an extensive panel of circulating sphingolipids (n=61).

Retinal vessel diameters, a novel surrogate of microvascular health that responds positively to exercise interventions, will be assessed pre- and post-intervention, and the brachial artery FMD, which reflects endothelial function as an early marker of atherosclerotic arterial damage.

Each participant will receive individualised, pre-packaged meals for the two days preceding blood sampling to minimise potential confounding. All participants will be fed to energy balance. To monitor diet adherence, participants will be instructed to return all non-consumed foods from the pre-packaged meals to the lab and take photos of additionally consumed foods for later analysis.

Inclusion Criteria:

• female or male sex,
• aged between 40 and 60 years,
• body mass index between 25.0 and 34.9 kg/m2,
• sedentary lifestyle, defined as not meeting the WHO guidelines on PA, i.e., at least 150 minutes of moderate-intensity aerobic PA per week as well as muscle-strengthening activities on two or more days per week,
• medical clearance for HIIT by a study physician (including vital sign evaluation, clinical examination, resting and exercise ECG),
• informed consent as documented by signature.

Exclusion Criteria:

• known pregnancy or breastfeeding,
• any current exercise-limiting musculoskeletal conditions of the lower limbs,
• any known current or chronic conditions limiting exhaustive exercise,
• known diabetes mellitus of any type,
• dyslipidaemia, if pharmaceutically treated,
• arterial hypertension ≥160/100 mmHg, pharmaceutically treated or not,
• any other known cardiovascular disease,
• known NASH,
• known macular degeneration, glaucoma, or high intraocular pressure (≥20 mm Hg),
• particular diet (vegetarian, vegan, lactose-free, gluten-free, or FODMAP-low diet (ferment-able oligosaccharides, disaccharides, monosaccharides, and polyols),
• intake of glucagon-like peptide-1 analogues, orlistat or any weight-loss drug,
• inability to follow the procedures of the study, e.g., due to linguistic or cognitive problems,
• concomitant involvement in another interventional trial or participation in another interventional trial in the last four weeks.

As the randomisation will take place after the baseline examination, participants, exercise scientists, and physicians supervising the intervention will be blinded for group allocation at baseline but not post-intervention.