## 3 Framework for In Vivo Cardiovascular Studies
### Human Considerations  
A shift in focus from flow phantoms to human participants brings about certain considerations for in vivo testing that may introduce unwanted variability into data quality. The human cardiovascular system is tightly regulated by the autonomic nervous system, involving beat-to-beat neurovascular regulation through the sympathetic and parasympathetic nervous systems [65; 66]. However, this regulation may offer unwanted sources of variability during HiFRUS studies, as only a few cardiac cycles can realistically be acquired per participant due to the high frame rate. Decisions on participant pre-visit instructions and study methodology should be made with consideration on how to minimize these confounders. For example, technical guidelines for evaluations of carotid-femoral pulse wave velocity and flow-mediated dilation have recommended that: participants refrain from food (\(\sim\)2-6 h), caffeine, smoking, and alcohol (\(\sim\)12 h) prior to measurement; testing should occur in a quiet, temperature-controlled room after 10 min of supine rest; and measurements be taken at the same time of day for longitudinal studies to account for circadian rhythm [67; 68; 69; 70; 71; 72; 73; 74]. While Table 2 lists recommendations for the average participant, any perturbations from the 'ambulatory' state of an individual should be considered; for example, asking an individual who habitually smokes to refrain from smoking may cause an equal amount of distress and undue sympathetic activation. As the novelty of HiFRUS data limits the available literature on the confounders of measurement variability, we suggest that the above basic study controls be considered for studies involving human participants, with the aim of reducing unwanted variability in the high-quality data. Medications are an important consideration when preparing to interpret the potentially clinically relevant information provided in HiFRUS examinations. As it is unethical and unsafe to ask participants to withhold all medication for certain studies, it is important to record and consider possible confounders when interpreting findings. Particular care should be taken with commonly prescribed medications for hypertension and heart disease, such as statins, beta-blockers, angiotensin-converting enzyme inhibitors, and diuretics, all of which result in measurable effects on cardiovascular function including reductions in heart rate, blood volume and arterial stiffness [67]. Further to the issue of beat-to-beat variability, additional considerations should be given to factors that may affect the ability of individual heart cycles to reflect'steady state' conditions of an individual or physiological state. Respiratory sinus arrhythmia is a known phenomenon in physiological monitoring, eliciting predictable fluctuations in heart rate and blood pressure [75]. Given that typical respiratory \begin{table}
\begin{tabular}{c c} \hline \hline
**Recommendation** & **Reason** \\ \hline Pre-visit instructions & \\ 2 h fasted & Altered sympathetic activation \\
6 h refrain from caffeine & Altered sympathetic activation \\
12 h refrain from smoking & Acute effects on vascular structure and function \\
12 h refrain from moderate-to-vigorous physical activity & Acute effects on vascular structure and function \\
12 h refrain from alcohol & Acute effects on vascular structure and function \\ Record of current medications & Various acute and chronic effects on the vasculature \\ \hline Participant preparation & \\ Assign unlinked participant ID & Ethical considerations for sensitive health information \\
10 min rest period & Altered sympathetic activation upon arrival to the lab \\ Resting heart rate recording & Detail of the hemodynamic environment \\ Resting blood pressure recording & Detail of the hemodynamic environment \\ Probe holder placement & Reduction of motion artifacts \\ Breath hold during acquisition & Reduction of motion artifacts \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Methodological considerations in cardiovascular human testing.