The Centre for Physical Activity Research (CFAS) is supported by TrygFonden (grants ID 101390 and ID 20045. JPH was supported by grants from Helsefonden and Rigshospitalet. During this work, RMGB was supported by a post.doc. grant from Rigshospitalet.

The study was evaluated by the Regional Ethical Committee of the Capital Region of Denmark (file no. H-21054272), who determined that this was a quality study. In accordance with Danish legislation, the study was thus approved locally by the internal Research and Quality Improvement Board at the Department of Clinical Physiology and Nuclear Medicine, Rigshospitalet (file no. KF-509-22). The study was performed according to the guidelines of the Declaration of Helsinki. All subjects provided oral and written informed consent prior to enrolment. Men and women, &#8805;18 years, were included in the study. Individuals with peripheral arterial disease, heart failure, neurological and musculoskeletal disease hindering KEE effort, and symptoms of disease within 2 weeks prior to the study, were excluded.
1. Setup of the participant
<ol>
	<li>Place the participant in the single-leg knee-extensor chair with the back of the participant resting against the chair (Supplementary Figure 1). Dress the participant in underwear that makes it possible to access the inguinal region with an ultrasound probe.</li>
	<li>Place three ECG electrodes (see Table of Materials) on the participant. Place the electrodes on the right side of the chest wall in the third intercostal space, on the left side in the third intercostal space, and on the left side in the eleventh intercostal space so that the electrodes are equidistant from the heart.</li>
	<li>Place the participant at a &#62;90-degree angle between the abdomen and thigh.</li>
	<li>Adjust the arm connecting the single-knee extensor chair to the flywheel to enable the participant to extend the knee fully.</li>
	<li>Tie the leg of the participant tightly to the pedal of the chair to avoid the usage of muscles in the lower part of the limb.</li>
	<li>Place a chair or a bench to stabilize the inactive leg. 
	NOTE: The angle of &#62;90 degrees is considered a minimum. Increasing the angle will open the inguinal area allowing better access to the femoral artery with the ultrasound probe. This approach is often used when subjects have abdominal adiposity that can interfere with scanning. 
	Adding resistance to the single-leg knee-extensor chair is done differently depending on the type and model and is thus not described in detail. Both absolute and relative intensity can be reported. In order to report relative intensity, perform a test to exhaustion on a preceding day.</li>
</ol>
2. Setup of the ultrasound apparatus
<ol>
	<li>Press the Turn on button.</li>
	<li>Press Patient to create a file where the examination will be saved. Move the cursor to &#34;new patient&#34; and press enter. Fill out the &#34;patient ID&#34; move the cursor to &#34;Create&#34; and press Enter (Supplementary Figure 2 and Supplementary Figure 3).</li>
	<li>Press Probe, choose the linear probe (9 MHz), and apply ultrasound gel (see Table of Materials) to the probe. 
	&#8203;NOTE: It is not possible to save the data from the participant without assigning a &#34;Patient ID&#34;. Assigning more data to this sheet is possible but not necessary for the examination to be performed.</li>
</ol>
3. Doppler ultrasound scan
<ol>
	<li>Operate the linear probe with the hand closest to the participant and place it in the inguinal region. Find the best arterial section for obtaining LBF measurements carefully. This is below the inguinal ligament and 3-4 cm above the bifurcation of the common femoral artery on a straight segment of the artery.</li>
	<li>Hold the probe perpendicular to the vessel. Press the 2D button and make a cross-sectional image of the common femoral artery (CFA).</li>
	<li>Optimize gain and depth, which are to be maintained throughout the experiment, to ensure that the artery is in the middle of the screen and that the blood is black. Turn the Gain button clockwise to increase gain and counterclockwise to decrease gain. Turn the Depth clockwise to increase the depth and counterclockwise to decrease it. 
	NOTE: Please see Supplementary Figure 2 and Supplementary Figure 3 for the localization of the buttons and Supplementary Figure 4 for an ultrasound image optimized with gain and depth.</li>
	<li>While in 2D mode, press Freeze once and scroll using the trackball to find an end-systolic image. Perform this under ECG-guidance by stopping the image at the end of the T-wave.</li>
	<li>Press Measure once and move the cursor to the superficial intimal layer of the artery, and press Enter. Move the cursor to the deep intimal layer of the artery and then press Enter to obtain the diameter at end-systole. The diameter will be shown in the top left corner.</li>
	<li>Press Freeze and turn the probe 90 degrees clockwise while keeping the artery in the middle of the screen and holding it parallel to the artery to create a longitudinal view. Press the pulse wave button PW and then press Measure. This will create a drop-down menu on the right side of the screen. Move the cursor to CFA and press enter.</li>
	<li>Move the cursor to &#34;Auto&#34; and press Enter. Move the cursor to &#34;Flow volume&#34; and press Enter. Move the cursor to &#34;Live,&#34; and press Enter to obtain the trace and finish by pressing Measure once.</li>
	<li>Obtain the velocity at the lowest possible insonation angle and always below 60 degree. Turn the Steer Angle button clockwise to decrease it and counterclockwise to increase it. Turn the Angle correction button to ensure the trace is obtained with the cursor being horizontal to the artery, as shown in Supplementary Figure 4.</li>
	<li>Press Sample vol. to adjust according to the width of the artery and keep clear of the walls of the artery. To decrease the sample size, press the left arrow. To increase the sample size, press the right arrow.</li>
	<li>Obtain the blood flow velocity trace with simultaneous 2D visualization of the artery and audio-visual blood velocity feedback. Ensure that the sound is on by turning the Sound button clockwise.</li>
	<li>Obtain the first trace during seated rest for a minimum of 30 s and press Image Store twice to save the trace. Then instruct the participant to keep a pace of 60 rounds per minute (RPM) during the test and to only use the quadriceps muscle to perform the leg extensions and keep the hamstring muscle relaxed. Keep the probe fixed during the entire experiment.</li>
	<li>Instruct the participant to keep a pace of 60 rounds per minute (RPM) at 0 W and only to use the quadriceps muscle to perform the leg extensions and keep the hamstring muscle relaxed. Keep the probe fixed during the entire experiment and press Image Store twice to save the trace.</li>
	<li>Add resistance and have the participant complete at least 150 s of exercise before obtaining the 30 s of trace and then press Image Store twice to save the trace.</li>
</ol>
4. Quantitation of blood flow
<ol>
	<li>Once all the images are obtained, press Review.</li>
	<li>Press Track Ball and move the cursor to the image of desire, and double-click on Enter.</li>
	<li>Once the desired trace appears, press Measure and move the cursor to &#34;Flow volume&#34; in the drop-down menu on the right side of the screen and press Enter.</li>
	<li>Move the cursor to the 2D ultrasound image, press Enter, then drag the cursor until it reaches the diameter measured during rest and press Enter again.</li>
	<li>Turn the Cursor Select button clockwise twice and choose the 30 s of trace that will be shown between two vertical lines by scrolling the trackball and pressing Enter.</li>
	<li>Calculate the LBF as the product of mean blood velocity (cm/s) and cross-sectional area of the femoral artery (cm2), which will be shown in the top left corner. 
	NOTE: Perform quality control prior to data analysis by visual inspection of the trace and exclude pulse waves affected by motion artifacts as well as irregular heartbeats. It is possible to adjust angle correction after completing the examination by turning the Angle Corr. button clockwise to decrease it and counterclockwise to increase it to ensure the cursor is horizontal to the artery.</li>
</ol>

Assessing Leg Blood Flow in Training Workloads

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This study assessed the reliability of Doppler ultrasound methodology for evaluating leg blood flow (LBF) during submaximal single-leg knee-extensor exercise in healthy participants. The results indicated high within-day reliability and acceptable between-day reliability, while inter-rater reliability was found to be unacceptable at rest and at 0 W.
Although probe removal between measurements appeared to have little impact, the difference in reliability between within-day and between-day measu...

Doppler ultrasound, introduced in the 1980s, has been extensively used to determine contracting muscle blood flow, particularly in the single-leg knee-extensor model, allowing measurement of blood flow in the common femoral artery (CFA) during small muscle mass activation1,2,3,4,5,6. Doppler ultrasound-based blood flow technology has provided valuable insights into vascular regulation in various populations, including healthy adults7,8, individuals with diabetes9, hypertension10, COPD11,12, and heart failure13,14.
One advantage of Doppler ultrasound is its non-invasiveness compared to other blood flow determination methods like thermodilution, and it can be combined with arterial and venous catheterization if necessary3,4,6,15. It also enables beat-to-beat blood flow velocity measurement, allowing for the detection of rapid changes16. However, Doppler ultrasound-based blood measurements have limitations, including difficulties in obtaining stable recordings during excessive limb movement at near-maximal exercise intensities and the requirement for ultrasound accessibility to the targeted blood vessel, excluding evaluations during ergometer bicycling15. Hence, the single-leg knee-extensor model is well-suited for LBF evaluation using Doppler ultrasound during dynamic exercise at submaximal intensities17, minimizing the influence of exercise-related heart and lung limitations and facilitating comparisons between healthy subjects and patients with cardio-pulmonary diseases11.
Despite being widely used, the between-day reliability of the single-leg knee-extensor model using Doppler ultrasound has not been investigated on a larger scale in recent decades, with prior studies involving small populations (n = 2)3,18,19,20.
This study aimed to investigate (1) the within-day test-retest reliability, (2) the between-day test-retest reliability, and (3) the inter-rater reliability of Doppler ultrasound for LBF evaluation during single-leg knee-extensor exercise at 0 W, 6 W, 12 W, and 18 W. The measurements were conducted in a clinically realistic scenario where the probe was removed between measurements. It is important to note that several intrinsic and extrinsic environmental factors known to influence LBF were not controlled during the measurements, which could introduce variability and affect reliability. Considering advancements in Doppler ultrasound technology and blood flow analysis software, we hypothesized that, even in an uncontrolled setting, acceptable within- and between-day reliability of LBF measurements could be achieved at all intensities when performed by the same sonographer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EKO GEL</td>
			<td>EKKOMED A7S</td>
			<td>DK-7500 Holstebro</td>
			<td></td>
		</tr>
		<tr>
			<td>RStudio, version 1.4.1717</td>
			<td>R Project for Statistical Computing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Saltin Chair</td>
			<td></td>
			<td></td>
			<td>This was built from an ergometer bike and a carseat owned by Professor Bengt Saltin. The steelconstruction was built from a specialist who custommade it.</td>
		</tr>
		<tr>
			<td>Ultrasound apparatus equipped with a linear probe (9 MHz, Logic E9)</td>
			<td>GE Healthcare</td>
			<td>Unknown</td>
			<td>GE Healthcare, Milwaukee, WI, USA</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Ultrasound gel</td>
		</tr>
	</tbody>
</table>

doppler ultrasound-based leg blood flow assessment during single-leg knee-extensor exercise in an uncontrolled setting

Doppler ultrasound has revolutionized the assessment of organ blood flow and is widely used in research and clinical settings. While Doppler ultrasound-based assessment of contracting leg muscle blood flow is common in human studies, the reliability of this method requires further investigation. Therefore, this study aimed to investigate the within-day test-retest, between-day test-retest, and inter-rater reliability of Doppler ultrasound for assessing leg blood flow during rest and graded single-leg knee-extensions (0 W, 6 W, 12 W, and 18 W), with the ultrasound probe being removed between measurements. The study included thirty healthy subjects (age: 33 &#177; 9.3, male/female: 14/16) who visited the laboratory on two different experimental days separated by 10 days. The study did not control for major confounders such as nutritional state, time of day, or hormonal status. Across different exercise intensities, the results demonstrated high within-day reliability with a coefficient of variation (CV) ranging from 4.0% to 4.3%, acceptable between-day reliability with a CV ranging from 10.1% to 20.2%, and inter-rater reliability with a CV ranging from 17.9% to 26.8%. Therefore, in a real-life clinical scenario where controlling various environmental factors is challenging, Doppler ultrasound can be used to determine leg blood flow during submaximal single-leg knee-extensor exercise with high within-day reliability and acceptable between-day reliability when performed by the same sonographer.

Author Spotlight: Assessing the Reliability of Doppler Ultrasound in Measuring Leg Blood Flow 

Doppler Ultrasound-Based Leg Blood Flow Assessment During Single-Leg Knee-Extensor Exercise in an Uncontrolled Setting

Participants 
From May 2022 to October 2022, a total of thirty healthy men and women were recruited to participate in the study. All participants had no history of cardiovascular, metabolic, or neurological diseases. They were not instructed to make any changes to their usual habits, including caffeine, alcohol, nicotine, vigorous exercise, or any other factors that could potentially impact vascular function.
Experimental procedures 
Participants rep...

Watch this Scientific Journal Video about Assessing the Reliability of Doppler Ultrasound in Measuring Leg Blood Flow at JoVE.com

This test-retest study evaluated leg blood flow measured by the Doppler ultrasound technique during single-leg knee-extensor exercise. The within-day, between-day, and inter-rater reliability of the method was investigated. The approach demonstrated high within-day and acceptable between-day reliability. However, the inter-rater reliability was unacceptably low during rest and at low workloads.

Doppler ultrasound has revolutionized the assessment of organ blood flow and is widely used in research and clinical settings. While Doppler ...

doppler-ultrasound-based-leg-blood-flow-assessment-during-single-leg

Research

JoVE Journal

Medicine

1.5K Views.  Copenhagen University Hospital. This test-retest study evaluated leg blood flow measured by the Doppler ultrasound technique during single-leg knee-extensor exercise. The within-day, between-day, and inter-rater reliability of the method was investigated. The approach demonstrated high within-day and acceptable between-day reliability. However, the inter-rater reliability was unacceptably low during rest and at low workloads.