Change in cerebral blood flow, as quantified non-invasively by the MRI perfusion method Arterial Spin Labeling (ASL), before and after application of intranasal insulin (160 IU).

Vascular health will be assessed by flow-mediated vasodilation (FMD, expressed as %), a non-invasive ultrasound technique that measures how well a blood vessel widens in response to increased blood flow. By briefly restricting blood flow in the arm (by means of a cuff on the fore arm) and then releasing it, investigators can see how effectively the brachial artery relaxes and dilates. This response reflects the function of the endothelium, the inner lining of blood vessels, and is considered an important indicator of cardiovascular health. The percentage indicates the relative increase in the brachial diameter compared to its baseline size.

The peripheral vascular function will be assessed by the carotid artery reactivity (CAR, expressed as %), a non-invasive technique that evaluates the ability of the carotid artery to dilate or constrict in response to specific physiological or pharmacological stimuli. The percentage indicates the relative change in the carotid artery's diameter compared to its baseline size. This method provides insight into endothelial function, vascular smooth muscle responsiveness, and overall arterial health, serving as an early marker for cardiovascular risk.

Arterial stiffness and central blood pressure will be assessed by pulse wave analysis (PWA). This non-invasive method records the shape of the pulse wave generated by each heartbeat, from the wrist or arm. From this signal, central blood pressure is expressed in millimeters of mercury (mmHg), while derived indices of arterial stiffness (such as the augmentation index, AIx) are expressed in percentages (%), representing the relative contribution of reflected waves to the overall pulse wave. By analyzing how the wave travels and reflects in the arteries, investigators can gain insight into arterial elasticity, vascular resistance, and the overall condition of the cardiovascular system.

Arterial stiffness will be assessed by pulse wave velocity (PWV, expressed in meters per second [m/s]). This non-invasive technique measures the speed at which the pressure wave created by the heartbeat travels through the arteries. Since stiffer arteries allow the wave to move faster, PWV provides a reliable indicator of vascular elasticity and cardiovascular risk.

The microcirculation will be assessed by measuring retinal microvascular calibers. Using non-invasive retinal imaging, the diameters of small blood vessels in the back of the eye are quantified. Because the retina offers a unique 'window' to the body's microvascular system, changes in these tiny vessel widths can reveal early signs of vascular dysfunction and give an indication about cardiometabolic health.

Blood pressure (systolic and diastolic) and heart rate will be assessed through both office measurements and 24-hour ambulatory monitoring. Office blood pressure provides standardized readings taken in a clinical setting, while ambulatory blood pressure monitoring records values at regular intervals throughout the day (every 15 minutes) and night (every 30 minutes) as individuals go about their daily lives. Together, these methods give a more complete picture of blood pressure patterns, capturing variability, nighttime values, and potential masked or white-coat hypertension.

Blood lipid levels will be assessed by measuring triacylglycerol (TAG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C). These standard blood tests provide important information about fat metabolism and cardiovascular risk.

Blood glucose will be measured from blood samples to determine the concentration of sugar in the bloodstream. Measuring blood glucose provides important information about metabolic health.

Insulin levels will be measured from blood samples to evaluate how the body regulates glucose. Measuring insulin provides insight into metabolic function and insulin sensitivity.

Markers for low-grade systemic inflammation (IL-6,TNF-alfa) will be assessed in the blood plasma to determine low-grade systemic inflammation.

Brain structure will be assessed using a structural MRI with an MPRAGE sequence. This high-resolution imaging technique provides detailed pictures of brain anatomy, allowing investigators to examine the size and shape of different brain regions.

Leucocytes will be isolated from EDTA blood. DNA will be isolated from these leucocytes according to standard procedures. DNA will be used for analyzing the presence of known SNPs in genes encoding proteins known to play a role in cholesterol metabolism.

Quality of life will be assessed using a 32-item questionnaire that covers multiple aspects of daily living and well-being. The questionnaire asks about physical health, emotional state, social relationships, and overall life satisfaction. By combining responses across these domains, it provides a broad picture of how individuals perceive and experience their quality of life.

Sleep characteristics will be assessed using the 10-item Pittsburgh Sleep Quality Index (PSQI). This standardized questionnaire asks about different aspects of sleep over the past month, including sleep duration, quality, and disturbances. By combining the responses, the PSQI provides an overall score that reflects both subjective sleep quality and potential sleep problems

Mood will be assessed using the Affect Grid, a simple self-report tool that measures two key dimensions of emotion: pleasure (ranging from positive to negative feelings) and arousal (ranging from calm to highly activated). Participants indicate their current state on a single-page grid, providing a quick and intuitive snapshot of their mood at that moment.

Stress will be assessed using the Perceived Stress Scale (PSS), a widely used questionnaire that asks about feelings and thoughts during the past month. The PSS captures how unpredictable, uncontrollable, and overloaded participants perceive their lives to be. Higher scores indicate greater levels of perceived stress, providing a useful measure of everyday psychological strain.

Weight will be determined in kilograms.

Length will be determined in meters.

Waist circumference in centimeters.

Hip circumference in centimeters.

Food intake will be assessed using the Food Frequency Questionnaire (FFQ), a standardized dietary survey that asks participants how often they consume a wide range of foods and beverages in the past month. This method provides an overview of habitual dietary patterns, nutrient intake, and overall diet quality, making it a useful tool for linking nutrition with health outcomes.

Cerebral perfusion will be assessed using Transcranial Doppler ultrasound, a non-invasive method that measures blood flow in the middle cerebral artery. During the procedure, continuous blood flow velocity (CBFv) is recorded through the skull using ultrasound waves. This approach provides valuable information about the cerebrovascular health and function.