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 cons...

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.

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			<td>EKO GEL</td>
			<td>EKKOMED A7S</td>
			<td>DK-7500 Holstebro</td>
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			<td>RStudio, version 1.4.1717</td>
			<td>R Project for Statistical Computing</td>
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			<td>Saltin Chair</td>
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			<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>
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			<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>
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			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Ultrasound gel</td>
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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

This method is widely recognized and applied in numerous clinical and research settings. The primary objective of this study was to assist the reproducibility of the method, thereby providing valuable insights into the measurement error. By doing so, we can say what is genuine physiological change and what is due to measurement error.Ultimately, this will enhance our understanding of this method. The primary obstacle lies in exploring physiological changes during various activities in everyday life, as conducting such investigation is feasible only under specific experimental setups. t has been over two decades since the last reliability study was conducted during single leg knee extensor exercise.Over this period, technology has significantly evolved, making it imperative to revisit this research area to gain valuable insight into the measurement era associated with the current method. Doppler ultrasound is non-invasive compared to other blood flow determination methods like Thermodilution, and it can be combined with arterial and venous catheterization if necessary. It also enables beat to beat blood flow velocity measurements so we could detect rapid changes.In the future, our main goal is to investigate the regulation of blood flow to contracting skeletal muscle in patients with chronic obstructive pulmonary disease.

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

Single-Leg Knee-Extensor Chair Setup and Ultrasound Apparatus Configuration for Leg Blood Flow Assessment

To begin, position the participant in the single leg knee extensor chair with their back resting against the chair. Place a chair or bench to stabilize the inactive leg. To ensure the electrodes are equidistant from the heart, fix one ECG electrode on the right side of the chest wall in the third intercostal space.Place the second one on the left side in the third intercostal space. Then place the third one on the left side in the 11th intercostal space. After electrode placement, position the participant at an angle greater than 90 degrees between the abdomen and thigh.Adjust the arm connecting the single knee extensor chair to the flywheel. This enables the participant to extend the knee fully. Tie the leg tightly to the pedal of the chair to avoid the usage of muscles in the lower part of the limb.Next, press the turn on button on the ultrasound apparatus. Press patient to create a new file for the examination. Move the cursor to new patient and press enter.Then fill out the patient ID.Move the cursor to create and press enter. Connect the lead to the electrodes. Press probe and choose the linear probe.Apply ultrasound gel to the probe.

Doppler Ultrasound Scan and Blood Flow Quantitation in Leg-Extension Exercises

To begin, place the probe on the inguinal region of the participant then find the optimal arterial section for leg blood flow or LBF measurements. Make a cross-sectional image of the common femoral artery. Turn the Gain button clockwise to increase gain and counterclockwise to decrease gain.Turn the Depth button clockwise to increase the depth and counterclockwise to decrease it. After adjustment, ensure that the artery is in the middle of the screen and the blood is black. In 2D mode, press Freeze once then scroll using the track ball to find an end-systolic image.Press Measure once and move the cursor to the superficial intimal layer of the artery then press Enter. Next, move the cursor to the deep intimal layer of the artery and press Enter to obtain the diameter at end-systole. Press Freeze then turn the probe 90 degrees clockwise while keeping the artery centered on the screen and probe parallel to the artery to create a longitudinal view.Press the PW button, followed by the Measure button. Move the cursor to auto and press Enter then click Flow Volume and press Enter. Move the cursor to Live and press Enter to obtain the trace and finish by pressing Measure once.To obtain the velocity at an insonation angle of less than 60 degrees. Turn the Steer Angle button clockwise to decrease it and counterclockwise to increase it. Turn the Angle Correction button to validate the trace captured with the cursor horizontal to the artery.Press Sample Volume to adjust the volume according to the width of the artery while avoiding contact with the artery wall. To decrease the sample size, press the left arrow and increase by pressing the right arrow. Next, turn the Sound button clockwise then obtain the blood flow velocity trace with simultaneous 2D visualization of the artery and audiovisual blood velocity feedback.Obtain the first trace during seated rest for 30 seconds, and press Image Store twice to save the trace. Then, instruct the participant to maintain a pace of 60 RPM during the test. Using the same conditions, record the trace data at a pace of 60 RPM and zero watts.Add resistance to the single leg knee extensor chair. Then, ask the participant to complete a minimum of 150 seconds of exercise before obtaining 30 seconds of trace. Press Image Store twice to save the trace.Once all the images are obtained, press Review, press track ball and move the cursor to the desired image then double click on Enter. Once the desired trace appears, press Measure. Then, from the dropdown menu, select Flow Volume and press Enter.Next, select 2D Ultrasound Image and press Enter. Then drag the cursor until it reaches the diameter measured during rest and press Enter again. Turn the Cursor Select button clockwise twice.Then, by scrolling the track ball and pressing Enter, choose a 30-second trace between two vertical lines. There were no statistically significant differences in the absolute LBF values between the Within Day or Between Day measurements. LBF increased progressively across the incremental workloads.The value ranges from 0.36 liter per minute at rest to 2.44 liter per minute during exercise at 18 watts. Bland Altman Plots of LBF measurements for Within Day reliability showed no outliers. In contrast, a few outliers were observed in the Between Day measurements, and several outliers were observed during the Interrater measurements.In test/retest reliability, the smallest real difference values were lower for Within Day measurements compared to the Between Day and Interrater measurements. Similarly, the coefficient of variation values were higher in the Between Day and Interrater measurements compared to the Within Day measurements. The intraclass correlation coefficient values showed that the reliability at all workloads during Within Day and Between Day were higher than 0.90.Conversely, the Interrater measurements yielded ICC values as low as 0.41.

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.

Assessing Leg Blood Flow in Training Workloads

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