This study adhered to the principles outlined in the Declaration of Helsinki regarding ethical standards for research involving human subjects. The study protocol was approved by the Swedish Ethical Review Authority (approval numbers 2020-00140, 2022-04442-02, and 2023-02922-02), and written informed consent was obtained from all participants. Participants had to be between 18 and 45 years old, generally healthy, free from previously known cardiovascular disease, and willing to participate voluntarily. Participants were excluded for blood pressures over 140/90, damaged or tattooed skin on fingertips or forearms, and pregnancy.

Following the informed consent procedure, 50 volunteers were screened (Fig. 1). Participants were randomly assigned to either of two provocation protocols: a stepwise escalation from 0 to − 70 mmHg (− 9.3 kPa) LBNP (“Stepwise”) and a direct application of − 70 mmHg (− 9.3 kPa) LBNP (“Direct”).

Schematic representation of inclusion and setup of the study. The Stepwise and Direct protocol are represented with flowcharts of data collection occasions, with the number of participants remaining and the male and female distribution on each occasion. The reason(s) for the participant drop-off is stated by; “Technical” in case technical difficulties of data acquiring/analyses resulted in participant drop-off, or “Completed” in case the drop-off is explained by participants exiting, and thereby completing the provocation protocol. Time scale is represented in minutes (min) and level of pressure provocation is represented in millimeters of mercury (mmHg)

The participants were free to end the provocation at any time. Other criteria for terminating the study included participants experiencing pre-syncope symptoms or displaying alterations in vital signs indicating imminent syncope, as determined by an experienced emergency medicine practitioner familiar with the LBNP model. The decision-making process involves weighing together classical subject-reported indicators of pre-syncope such as sudden dryness of the mouth, diaphoresis, nausea and sudden-onset headache. Further, this assessment was assisted by continuously monitoring the trend of vital signs, where pre-syncope is typically characterized by a sudden drop in blood pressure (increasingly negative slope of the curve, rather than an absolute value), and tachycardia. Additionally, the study would conclude upon reaching the protocol’s maximum duration of 60 min at − 70 mmHg (− 9.3 kPa).

Prior to the initiation of the experiment, all participants rested for at least 5 min in a supine position in the inactivated LBNP chamber at atmospheric pressure. Baseline measurements were gathered for all included parameters [CR time, systolic and diastolic blood pressure, mean arterial pressure (MAP), heart rate, cardiac output, and systemic vascular resistance (SVR)] before the LBNP provocation started.

Lower body negative pressure (LBNP) is a non-invasive and rigorously validated method used to mimic hemorrhage [1, 4]. By decreasing the air pressure around the lower extremities of a human volunteer, LBNP induces central hypovolemia, causing blood to pool in the lower limbs and diverting blood volume away from the thoracic region, thereby replicating the effects of blood loss [1, 4]. This results in reduced venous return to the heart, leading to decreased cardiac output and arterial pressure. Consequently, the body initiates compensatory mechanisms, including increased heart rate, to sustain blood flow to vital organs [17, 18]. At the microcirculatory level, the response has previously been shown to involve a pattern of selective vasoconstriction and vasodilation bringing about a redistribution of the diminished cardiac output in rats [19]. In the cutaneous microcirculation, peripheral resistance rises, and the density of perfused capillaries diminishes, thereby reducing oxygen delivery [17, 20]. This model serves as a valuable tool for investigating physiological responses and adaptations to hypovolemia [1, 4, 17, 18].

During the experiment, participants were subject to pressures between 0 and − 70 mmHg (− 9.3 kPa) below atmospheric pressure, on their lower body (Fig. 1).

Specifically, the LBNP provocation for the Stepwise protocol started at − 20 mmHg (− 2.7 kPa, under atmospheric pressure). The negative pressure was maintained for 5 min before de-escalating in decrements of − 10 mmHg (− 1.3 kPa) until finally reaching − 70 mmHg (− 9.3 kPa, under atmospheric pressure) or if any of the stopping criteria were met. Two CR time measurements were made: one in the beginning and one within the last minute of each pressure, before increasing the negative pressure level. This was the procedure for each pressure level until the second measurement at − 70 mmHg (− 9.3 kPa), where subsequent measurements were made at intervals of 3 min instead, until maximum 60 min.

The Direct protocol started at an immediate pressure of − 70 mmHg (− 9.3 kPa) relative to atmospheric pressure. This pressure was maintained until any stopping criteria were met, or for a maximum of 60 min. CR time measurements were taken just after the negative pressure stabilized, and subsequent measurements were taken at 3-min intervals.

Post-provocation, the participant remained in a supine position in the experimental setup for at least 10 min to ensure recession of any pre-syncope symptoms and normalization of blood pressures and heart rate. During this period, post-provocation CR time measurements were taken. Post-provocation CR time measurements were done at 5 and 10 min in the Stepwise protocol and at 3, 6 and 9 min in the Direct protocol.

CR tests were made at the right-hand index fingertip (pulp) and were standardized using a 10 N dynamometer (TickIt 10 N, Hands-On-Science, Järfälla, Sweden). The dynamometer was fitted with a 1.77-cm2 round plastic cap to ensure an evenly distributed pressure (0.58 kg/cm2) over a standardized area. The blanching pressure was applied for 5 s. CR dynamics were assessed using a polarized reflectance imaging system.

The principles for the polarized reflectance imaging system have been described in detail elsewhere [21]. In short, it is a digital camera-based system that utilizes polarized white light along with a cross-polarized detector to minimize surface glare. The system uses the varying absorption properties of red blood cells and surrounding tissue to derive a value indicative of the concentration of red blood cells in the superficial skin layers. Unlike the surrounding tissue, moving red blood cells exhibit significant light absorption in the green wavelength range (approximately 500–600 nm) and light reflection in the red wavelength range (approximately 600–700 nm). This spectral difference enables visible light spectroscopy through an algorithm that separates the red, green, and blue color matrices and subtracts the green component from the red in each pixel, yielding an output matrix reflecting local red blood cell concentration. Set to video mode, it provides objective means of assessing CR dynamics.

In the present study, the system was set to record at 50 frames per second, at a resolution of 1920 × 1080 pixels.

Systolic and diastolic blood pressure, MAP, SVR and cardiac output were monitored with 1-s resolution using a Finapres (Finapres Finometer Midi, Finapres Medical Systems B.V, The Netherlands). The Finapres sensor was situated on the left-hand middle finger and monitored systolic blood pressure (mmHg), diastolic blood pressure (mmHg), MAP (mmHg), heart rate (beats/min) SVR (Dynes/s/cm−5) and cardiac output (L/min).

Brachial blood pressure (systolic, diastolic and MAP) and heart rate (beats/min) were also monitored with a Philips IntelliVue MP-30 (IntelliVue MP-30, Philips Electronics North America Corp., United States) as a back-up system to the Finapres. Medical staff were always present on site to evaluate the subject’s health and vital signs.

Statistical tests were performed using GraphPad Prism version 10.0.2 for Windows (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com).

The parameters heart rate, cardiac output, SVR and CR time were all compared “Baseline” to “Maximum Provocation” (or “Max Prov.”). With an exception for CR time, Baseline was defined as the average of a 5-min period before the provocation, and the Maximum Provocation was defined as the average of the last measurement period before the provocation ended. For CR time, Baseline was defined as the CR test made before the provocation, and the Maximum Provocation was defined as the last CR test made before the provocation ended.

The blood pressure parameters: systolic, diastolic and MAP, were all compared “Baseline” to “Lowest”. For the blood pressure parameters, Baseline was defined as the average of a 5-min period before the provocation, and Lowest was defined as 30 s before the provocation ended.

The distribution of all data was tested with the Shapiro–Wilk normality test. The Wilcoxon matched-pairs signed rank test was used to analyze if there were significant changes in the investigated parameters, and the Mann–Whitney test was used testing significant differences between groups.