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Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test

Published: April 27, 2016 doi: 10.3791/54011


The flow mediated dilation (FMD) test is the most commonly utilized, non-invasive, ultrasound assessment of endothelial function in humans. Although the FMD test has been related with the prediction of future cardiovascular disease and events, it is a physiological assessment with many inherent confounding factors that need to be considered.


Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a pathological condition characterized mainly by a disruption in the balance between vasodilator and vasoconstrictor substances and is proposed to play an important role in the development of atherosclerotic cardiovascular disease. Therefore, a precise evaluation of endothelial function in humans represents an important tool that could help better understand the etiology of multiple cardio-centric pathologies.

Over the past twenty-five years, many methodological approaches have been developed to provide an assessment of endothelial function in humans. Introduced in 1989, the FMD test incorporates a forearm occlusion and subsequent reactive hyperemia that promotes nitric oxide production and vasodilation of the brachial artery. The FMD test is now the most widely utilized, non-invasive, ultrasonic assessment of endothelial function in humans and has been associated with future cardiovascular events.

Although the FMD test could have clinical utility, it is a physiological assessment that has inherited several confounding factors that need to be considered. This article describes a standardized protocol for determining FMD including the recommended methodology to help minimize the physiological and technical issues and improve the precision and reproducibility of the assessment.


Cardiovascular disease is the leading cause of morbidity and mortality worldwide. Dysfunction of the vascular endothelium represents an initial phase toward the development of multiple vascular-related diseases1. Hence, an accurate assessment of endothelial function in humans represents an important technique that could help in understanding the etiology of multiple cardiovascular pathologies, with the ultimate goal of improving the efficacy of the treatment and prevention of disease.


The endothelium is a monolayer of cells that synthesizes numerous vasoactive substances, such as nitric oxide (NO), prostacyclins, endothelins, endothelial cell growth factor, interleukins, and plasminogen inhibitors2. Such factors contribute to the endothelium's function to regulate blood fluidity, vascular tone, platelet aggregation, permeability of plasma components and vessel wall inflammation2-4. Additionally, NO plays a key anti-atherogenic role in promoting vasodilation and maintaining endothelial integrity. NO regulates vessel tone and diameter through controlling the equilibrium between the delivery of oxygen to the tissues and their metabolic demand3,5. There are multiple endogenous, exogenous, and mechanical stimulator factors that induce endothelial NO synthase (eNOS) which synthesizes NO from L-arginine6,7. The most notable mechanical stimulus is shear stress. Wall shear stress contributes to greater activation of eNOS, resulting in NO production and subsequent smooth muscle relaxation4. For that reason the decrease in NO bioavailability is often used as a measure of endothelial dysfunction8.

Endothelium dysfunction

The imbalance between vasodilator and vasoconstrictor factors leads to a dysfunctional endothelium2. In addition, the release of inflammatory mediators and altered local shear forces may enhance the synthesis of endothelial derived reactive oxygen species (ROS). This upregulation in redox signaling not only modifies the integrity of the endothelium and reduces the synthesis of NO9, it can uncouple eNOS resulting in direct production of additional free radicals. Ultimately, this amelioration in NO bioavailability promotes vasoconstriction, vascular stiffness, and reduced arterial distensibility4.

The degree of dysfunction of the endothelium has been related with the severity of several pathologies such as hypertension10, atherosclerosis11, ischemic stroke12, diabetes13, preeclampsia14 or kidney diseases15 among others. Hence, there is vast interest to not only evaluate changes in endothelial function over time, but also following therapeutic interventions. Different methods have been used for the clinical assessment of endothelial function both invasively (cardiac catheterization and venous occlusion plethysmography3,16) and non-invasively (flow mediated dilation, radial artery tonometry and pulse contour analysis4,17,18) in coronary and peripheral circulations19.

Flow-mediated dilation

Flow mediated dilation (FMD) is a non-invasive, ultrasonic evaluation of endothelial function and has been correlated with the development of vascular health problems. Since its inception in 198920, FMD has been widely utilized as a reliable, in vivo method to evaluate predominately NO-mediated endothelial function in humans19,21,22. Indeed, the brachial artery FMD test has been associated with other invasive techniques23 and numerous investigations have described a strong inverse relationship between FMD and cardiovascular injury24,25 such that individuals with more vascular pathology exhibit a lower FMD25. Accordingly, these data emphasize the prognostic information that this technique can provide as it relates to future cardiovascular disease in asymptomatic subjects26-30.

During the FMD test, the diameters of the brachial artery are continuously measured at baseline and after the release of a circulatory arrest of the forearm. Upon cuff release, the induced-reactive hyperemia promotes an increase in shear stress mediated NO release and subsequent vasodilation19,31. FMD is expressed as the percent increase in arterial diameter following the release of the cuff compared with the diameter at baseline (FMD%).

Despite the increasing clinical interest in this technique, the FMD test is a physiological assessment and therefore, several variables need to be considered in order to conduct a precise assessment of endothelial function in humans. This article describes a standardized protocol and the recommended methodology to minimize the technical and biological issues to help improve the accuracy, reproducibility and interpretation of the FMD test.

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NOTE: The following FMD procedure is routinely conducted during vascular assessment studies in the Laboratory of Integrative Vascular and Exercise Physiology (LIVEP). All procedures followed the principles of the Declaration of Helsinki and were approved by the Institutional Review Board at Georgia Regents University. All participants were informed of the objectives and possible risks of the technique before written consent for participation was obtained. Figure 1 illustrates a schematic summary of the essential elements that should be considered for the ultrasound assessment of brachial artery FMD.

1. Subject Preparation (Prior to Arrival)

  1. Confirm that the participant has abstained from practicing exercise (≥12 hr), caffeine (≥12 hr), smoking or smoke exposure (≥12 hr), vitamin supplementation (>72 hr) and any medication (≥4 hr half-lives of the drug, non-steroidal anti-inflammatory agents for 1 day and aspirins for 3 days).
  2. Ensure that the participant is under fasting conditions or has only consumed low-fat meals4 prior to testing.
  3. When testing premenopausal women, it is suggested to conduct the FMD protocol during the menses phase of the menstrual cycle to limit the impact of endogenous estrogens and progesterones8,32,33.

2. Subject Preparation (Upon Arrival)

  1. Prior to measurement acquisition, verify that the subject is resting in a supine position in a quiet, temperature-controlled (22 °C to 24 °C) room for approximately 20 min to achieve a hemodynamic steady state.
  2. Attach a 3-lead ECG in the standard limb lead II position. Using American standard instrumentation, place the white/negative polarity lead just below the clavicle on the right shoulder. Connect the black/ dual polarity lead below the left clavicle near the shoulder and connect the red/positive polarity lead below the left pectoral muscle in the lateral base of the chest.
  3. Extend the subject's arm laterally at about 80° of shoulder abduction and secure the distal forearm in a vacuum packed pillow to maintain accurate position of the arm during the measurement (Figure 2).
  4. Place the forearm cuff immediately distal to the medial epicondyle and ensure that nothing is touching the cuff, including the table below (Figure 2).

3. Baseline Measurements

  1. Mapping the Brachial Artery with the Ultrasound:
    1. While holding the probe with the hand, position it cross-sectionally and start scanning the inner side of the upper arm beginning at the insertion of the bicep and proceeding proximally.
    2. Within B-mode (gray-scale), identify the brachial artery and collateral vessels and use color flow (CF) mode to help confirm the location of the artery. Interpret the color and pulsatility carefully considering the direction of the transducer to ensure assessment of the artery and not the vein.
      NOTE: With the probe indicator facing the head, red color means flow toward the transducer (arterial flow), while blue means flow away (venous flow).
  2. Identification of Brachial Artery:
    1. After finding the brachial artery, rotate the probe 90° to scan the arm longitudinally. Obtain the image between 2 to 10 cm above the antecubital fossa.
    2. Identify anatomical landmarks such as veins and fascial planes for multiple assessments in the same subject (Figure 3).
  3. Securing the Probe:
    1. Secure the probe in the stereotactic probe holder. Confirm the probe is appropriately fixed to avoid excessive movements. With the probe secured in the holder, ensure that the image is as good as the image that was obtained manually without the holder.
  4. Optimizing the Resolution of the Image:
    1. Optimize the image using the time gain controls (TGC's) with the probe secured.
      NOTE: An optimal image is achieved when the clearest B-mode image from the anterior and posterior intimal interfaces between the lumen and vessel wall is obtained.
    2. Have the technician manually adjust the gain, focal points, dynamic range, and harmonics to get a clear and defined image of the near and far walls of the endothelium.
  5. Duplex Doppler Mode:
    1. Following B-mode acquisition, proceed to duplex scanning in the pulsed Doppler mode.
    2. Use a heel to toe approach with the probe inside the holder by rocking the transducer up on one end more than the other to adjust the brachial artery image and obtain an angle of insonation of 60°.
  6. Baseline Acquisition:
    1. Obtain a satisfactory B-mode image that identifies the endothelial layers with clear intima-intima walls of the artery. Ensure that the Doppler signal appears sharp and clear sound with no muffles.
    2. Reset the ultrasound CINE loop by freezing and unfreezing the image. Press F1 to begin recording data on image software. Record baseline data for at least 30 sec. Analyze the average diameter and blood velocity for 30 sec to represent baseline values. Note: Different ultrasounds and software set-ups may require different sequences to obtain the required action.

4. Vascular Occlusion Measurements

  1. Forearm Occlusion:
    1. Rapidly inflate the forearm occlusion cuff, using compressed air, to supra-systolic pressures (250 mm Hg) for 5 min to induce arterial occlusion.
    2. After 4 min and 30 sec of forearm occlusion, begin acquiring data.
      NOTE: Occlusion measurements will be represented by the last 30 sec of occlusion.

5. Reactive Hyperemia (Post Cuff Release) Measurements

  1. Continuing to Acquire Data from Pre-cuff Release:
    1. Deflate the cuff at 5 min.
    2. Maintain recording for two minutes following cuff release.
  2. Following 2 min of post cuff release recording, stop and save the recordings. The highest 5 sec averaged interval throughout the 2 min post-occlusion collection period will be used to represent the peak hyperemic diameter.

6. Analysis of the Results: Edge Detection and Wall Tracking

  1. Due to the complexity of FMD analysis, use edge-detection and wall-tracking software throughout FMD testing for higher reproducibility according to manufacturer's instructions.
    NOTE: This offline analysis is less operator dependent than the manual assessment and therefore improves the accuracy of the FMD data4,34-36. In addition, this off-line analysis system also permits the synchronization with the ECG for the identification of end-diastolic arterial diameters, avoiding distortion of pulse-related changes in diameter4. It should be noted that, although the use of ECG is endorsed to minimize pulsation variability, it is also possible to perform the FMD protocol without ECG gaiting37. Although not recommended, if edge computer-assisted analysis is unavailable, careful manual assessment of both diameter and velocities should be collected36.
  2. For the evaluation of vessel diameters, it is necessary to visually inspect each frame to determine the best placement of the ultrasonic calipers along the B-mode image38.
    NOTE: Regardless of data analysis method, it is recommended to collect diameter and velocity data every 4 sec during the first 20 sec of reactive hyperemia and every 5 sec for the remaining post occlusion period4.

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Representative Results

Baseline characteristics from an apparently healthy cohort group are presented in Table 1. The most common variables of FMD testing conducted in the Laboratory of Integrative Vascular and Exercise Physiology (LIVEP) are presented in Table 2. The following variables are considered the main FMD parameters to analyze by the published FMD tutorial4 and guidelines36.

Baseline and peak diameter

Following an adequate acclimation phase, the average of at least 10 cardiac cycles with blood velocities over a time period of 10 to 30 sec39 should be used to represent baseline diameter. In addition, peak diameter, the maximal dilation following cuff release, should be calculated based on the highest 5 sec average over the two-minute post occlusion collection period4.

FMD response

The FMD response is represented by the maximal change in brachial artery diameter following the release of the forearm cuff relative to the baseline brachial artery diameter measured at rest. Hence, the determination of the FMD response is calculated according to the following equation:

Equation 1

The magnitude of the FMD response is directly proportional to shear stress and critically dependent on endothelial integrity, viscosity of blood, and blood velocity36,40. It has been observed that the maximum FMD is attained over a time window between 45 to 90 sec, although peak vasodilation itself may continue up to 180 sec post cuff deflation41.

Shear rate

Shear stress has been described as the parallel, frictional force exerted by the blood on the intima surfaces and as the primary stimulus for the FMD response42. Shear Stress can be calculated as a product of velocity and viscosity divided by the vessel diameter. However, a simpler index of shear stress is shear rate, which is calculated from simultaneous measurements of blood velocity and brachial artery diameter with the following equation:

Equation 2

In addition, cumulative shear rate (area under the curve, AUC; sec-1) has to also be considered since it reflects the delay between the peak shear and the peak diameter8. It is calculated based on the trapezoidal rule, every 4 sec for the first 20 sec following cuff release, and for the rest of the data collection period, every 5 sec43.

Normalization of FMD (FMD/Shear)

Given the reliance between shear stress and FMD, and keeping in mind the inter-subject variability of the reactive hyperemia response, it has been suggested to normalize the FMD response with shear stress44,45. Although there is no consensus on how to properly normalize FMD for shear, dividing FMD by shear rate controls the influence of different shear profiles in the FMD response and offers additional insight into the stimulus/response mechanism of brachial artery vasodilation39,46. However, it should be noted that, there is a rising awareness and temporary acceptance of this normalization method because it is only valid in certain conditions36. Another proposed way to normalize FMD and improve the sensitivity and reliability of the test is to express the data as a dose-response curve, where shear is related to the magnitude of arterial dilation47. Furthermore, the use of allometric scaling to normalize FMD and control for the impact that baseline diameters may have on the FMD response has been proposed48.

Time-to-peak vasodilation

Owing to the diverse responses in the time course of FMD described amongst various populations, determining the time-to-peak (TTP) vasodilation when analyzing FMD has become important49,50. However, it should be noted that TTP is partially NO independent, and may not be an appropriate FMD parameter to be used alone to represent endothelial health51.

Figure 4 illustrates the diameter and velocity response of an FMD test in a representative individual. Reactive hyperemia elicits a peak velocity which, after a short delay, is followed by increase in diameter.

Figure 1
Figure 1: Illustration of the standardized procedure to conduct a brachial artery FMD test. For an accurate and a reliable FMD test, it is essential to have appropriate ultrasound equipment as well as adequate preparation of the subject prior to performing the technique. Once the participant is in a resting state, the acquisition of baseline data can be performed. After five minutes of occlusion, the cuff is released creating a reactive hyperemic response that elicits shear stress on the endothelium. Finally, analysis of the results using an edge-detection and wall-tracking software is recommended. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Representation of a subject prepared for the brachial artery FMD test. The subject's arm is extended laterally and secured in a vacuum packed pillow. The forearm cuff is placed immediately distal to the medial epicondyle. The ultrasound transducer is secured in the holder and placed above the insertion of the biceps to acquire data of the brachial artery.

Figure 3
Figure 3: Identification of anatomical landmarks for repetitive assessments in the same subject. Figures 2A, 2B, 2C and 2D illustrate the baseline assessment of the brachial artery in the same individual on four different days. Arrows identify the anatomical landmark used to image the artery on each separate day. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Individual representation of a typical velocity and diameter response that is observed during the FMD test. The figure illustrates an initial baseline (BL) period of 30 sec, the last 30 sec of vascular occlusion (OCC), and the reactive hyperemia response (120 sec) following the forearm cuff release. The solid line represents the diameter response and the dotted line represents blood velocity throughout the FMD technique. Please click here to view a larger version of this figure.

n 62
Male/Female 31/31
Age (yrs) 32 ± 2
Height (cm) 167 ± 2
Weight (kg) 66.1 ± 2.2
BMI (kg/m2) 22.8 ± 0.7
SBP (mm Hg) 115 ± 2
DBP (mm Hg) 68 ± 1
FEV1 predicted (%) 101.2 ± 1.8
Glucose (mg/dl) 88 ± 1
Total cholesterol (mg/dl) 162 ± 5
HDL cholesterol (mg/dl) 57 ± 2
LDL cholesterol (mg/dl) 93 ± 5
Triglycerides (mg/dl) 77 ± 5
Hemoglobin (g/dl) 14.7 ± 0.3
Hematocrit (%) 43.4 ± 0.7

Table 1: Characteristics and blood chemistry of a healthy subject cohort. Data are expressed as mean ± SEM. Body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), Forced expiratory volume in one second (FEV1), high-density lipoproteins (HDL), low-density lipoproteins (LDL).

Variable n = 62
Baseline diameter (cm) 0.322 ± 0.009
Peak diameter (cm) 0.343 ± 0.009
FMD (%) 6.7 ± 0.4
FMD absolute change (cm) 0.021 ± 0.001
Shear rate (sec-1, AUC) 46,607 ± 2,940
FMD/Shear ( %/sec-1 AUC) 0.16 ± 0.01
Time-to-peak vasodilation (sec) 44 ± 2

Table 2: Brachial artery FMD variables from an apparently healthy cohort. Data are expressed as mean ± SEM.

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Introduced in 198920, the FMD test has been widely used in humans as a non-invasive measure of endothelial function. FMD has not only been shown to predict future vascular-related disease risk19,52,53, lower FMD values have been show to strongly correlate with cardiovascular impairments24,25,54. Although there are other techniques to assess endothelial function, both invasively (coronary angiography) and non-invasively (venous plethysmography and finger plethysmography), FMD has been the most widely used due to its non-intrusiveness and rapid evaluation of peripheral artery function23.

The FMD test incorporates a transient forearm occlusion which induces reactive hyperemia and subsequent shear stress2,4. This enhancement of shear stress results in a local increase of NO production from endothelium-derived NO synthase31,55, that diffuses through the vessel wall thereby inducing smooth muscle relaxation and subsequent, vasodilation31. It is generally accepted that 5 min occlusion periods are predominately mediated by NO, while the increase in blood flow after longer periods of occlusion may involve ischemia-induced vasodilators other than NO56.

Methodological considerations for FMD assessment

The non-invasiveness of the brachial artery FMD test has increased the interest in this technique. However, it is worth noting that there are practical challenges and methodological considerations that affect the physiological and technical stability of this procedure, restricting its clinical use57. Specifically, conducting the FMD test requires a substantial initial investment to purchase the necessary equipment (i.e. ultrasound, rapid cuff inflator, and the analysis software). In addition, a highly skilled and trained person/sonographer that understands the physiology of the FMD test is needed to perform the FMD test. In terms of methodology, it is important to consider that there are many different methodological approaches of the FMD test without any standardization. Consequently, normative data is different from lab to lab making it difficult to define "true" abnormalities in endothelial function. In addition, many confounding factors can affect the FMD test, and therefore a comprehensive understanding of the basal state of the person being tested is necessary to rule out false negative values. Nonetheless, conforming to the updated FMD test recommendations for a high precision of the technique and reduce FMD variability can be achieved.

Ultrasound technology

Up to date ultrasound technology is essential. The use of simultaneous acquisition of B-mode diameter and pulse-wave Doppler velocity has been recommended for a more rigorous detection of diameter changes4 and an accurate calculation of shear rate. The absence of duplex mode technology could be linked with some of the results which described similar degree of vasodilation after a 5 and a 10 min occlusion period8,58,59, whereas more recently studies using duplex mode have described how prolonged periods of ischemia are related with greater reperfusion33,60.

Moreover, to improve the precision of the FMD test, it is important to achieve an appropriate angle of insonation between the Doppler beam and the alignment of the artery. It is recommended to obtain an insonation angle of ≤60° to achieve the balance of an adequate image quality and reduce the level of velocity error4,36. It is also worth noting that at least a 10 MHz linear array transducer, with the use of a stereotactic clamp61,62, is recommended to obtain high quality B-mode images4.

Cuff position

The placement of the cuff has been examined in detail in many different studies8,63,64 since its size and its position can not only contribute to changes in the shear stress stimulus, but may also impact the mechanisms contributing to the FMD response41,63,65. It is recommended to place the occlusion cuff on the forearm, distal to the ultrasound probe, to induce an endothelium-dependent vasodilation36.


To achieve an accurate FMD, an automatic software analysis system with edge detection algorithm is highly recommended4. Edge detection relies on the determination of a precise baseline diameter, which is essential and the basis for the calculation of a valid FMD. An average of at least 10 cardiac cycles (or 30 sec) are needed to represent baseline diameter, whereas the peak diameter response should be calculated using a 5 sec average4.

In conclusion, the assessment of endothelial function has been considered an important evaluation for the advancement of several pathologies due to the multiple regulatory functions that the endothelium plays in the body. The FMD test represents a non-invasive tool that predicts cardiovascular disease and events; however, the high-technical demand and operator-dependent requirements have limited its broader application. Accordingly, there is increasing interested in evaluating endothelial function in humans to provide valuable clinical and physiological information. This article documents a series of recommendations to improve the accuracy in the brachial artery assessment of FMD.

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The authors have nothing to disclose.


The authors would like to thank the many subjects and patients who have participated in our studies in which we have evaluated endothelial function using the FMD test.


Name Company Catalog Number Comments
Doppler ultrasound GE Medical Systems  Logiq 7 Essential to include Duplex mode for simultaneous acquisition of B-mode and Doppler
Electrocardiographic (ECG) gating  Accusync Medical Research Accusync 72
12-MHz Linear array transducer  GE Medical Systems 11L-D A high-resolution linear array probe is essential
Forearm occlusion cuff  D.E. Hokanson SC5 5 cm x 84 cm
Ultrasound transmission gel  Parker 01-08
Rapid cuff inflator D.E. Hokanson E-20 AG101
Sterotactic-probe holder Flexabar  18047 Magnetic base fine adjustor
Edge detection analysis software Medical Imaging Applications Brachial Analyzer 5



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Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test
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Rodriguez-Miguelez, P., Seigler, N., More

Rodriguez-Miguelez, P., Seigler, N., Harris, R. A. Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test. J. Vis. Exp. (110), e54011, doi:10.3791/54011 (2016).

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