## 4 Research Example: Neurovascular Control and Complex Blood Flow  
The above general recommendations for in vivo investigations provide a general framework under which the acquisition and management of human data should be optimally performed in cardiovascular research. In order to supplement these considerations, below we present an example of a simple and realistic study design for a basic science research question, describing each step in the design process from research question to data acquisition. The most attractive element of HiFRUS for a cardiovascular physiologist is the unprecedented quantity and quality of information that can non-invasively be assessed from the conduit arteries. High temporal and spatial sensitivity of complex blood flow patterns may offer a unique view of traditional cardiovascular techniques that have otherwise been well established in the field. For example, the cold pressor test (CPT) is a well-documented assessment of neurovascular reactivity [79; 80], which has a history of central and peripheral responder sub-types [81], and documented effects on intra- and extra-cranial blood flow [82; 83]. The CPT is highlighted by its simple protocol: submersion of either the hand or foot into an ice bath (\(\sim\)2-4 \({}^{\circ}\)C) for a duration of \(\sim\)2 min. This stimulus elicits a rapid neurovascular response, characterized by \(\alpha\)-adrenergic peripheral vasoconstriction causing slight increases in heart rate (i.e., +5 to +10 beats per minute) and moderate increases in mean arterial pressure (i.e., +15 to +25 mmHg). While recent studies have reported increases in common carotid, internal carotid, and middle cerebral artery blood velocity with the CPT [82; 83], it may be of value to characterize the complex flow patterns at the carotid bifurcation to further detail the known hemodynamics of the CPT, as well as to perhaps develop an easily accessible reactivity test for carotid bifurcation jets and recirculation zones under challenge conditions. From this knowledge gap in a well-established cardiovascular technique, we can design an acute interventional within-subject human research question that directly uses the novel capabilities of HiFRUS with a priori hypotheses: does the CPT elicit changes in carotid bifurcation flow jet velocity or recirculation zones as measured by HiFRUS? Figure 2 and Table 1 detail the methodological and technical design elements that we would employ for such a study. Young healthy adults are the ideal participants for this design, as we would like to test a basic science question in a controlled system, ideally by manipulating only the variables  
of interest without considering confounding pathology. After institutional safety and ethics approval, participants would be recruited and consented for testing in the lab. To ensure quality data, we suggest that potential participants be screened for appropriate scanning windows for appropriate orientation of the carotid bifurcation (i.e., at minimum, in-plane visualization of the carotid bulb and internal carotid artery). This exclusion criteria limits the generalizability of the experimental findings, but we must acknowledge the variability in carotid geometry in the general population using 2D ultrasound [84; 85]. To limit confounding factors for measurement variability, we would ask participants to arrive at the lab having fasted for two hours, and having refrained from moderate-to-vigorous physical activity, smoking, and alcohol in the 12 h prior to assessments (Table 2). During the testing protocol, participants would rest supine for at least ten minutes prior to data collection to standardize the hemodynamic environment (i.e., heart rate and blood pressure). For this particular research question, it would be valuable to gate analysis to the electrocardiogram (ECG) trace, in order to account for some beat-to-beat variability in blood flow, as well as to assist with aligning secondary data acquisition such as beat-to-beat blood pressure finger plethysmography or transcranial Doppler signals.