Subject preparation
fasting state (>6h) no smoking or any tobacco consumption at least 6h before study no exercise or food/beverages that contain alcohol, caffeine or are rich in polyphenols (cocoa, tea, fruit juices for >12h no vitamins for at least 72h withheld vasoactive medications on the morning of the study if possible and carefully note use and timing of any drugs no exercise >12h prior to test quiet, temperature-controlled room in females, repetitive studies should be made at the same time of menstrual cycle (ideally assess on days 1–7 of the menstrual cycle) rest for at least 10 minutes prior to measurements supine position arm resting comfortable with cradle support with the imaged artery at the heart level test should be performed at the same time of the day (especially if multiple tests are performed)
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Sphygmomanometer probe position and cuff occlusion time
placement of the cuff 1–2 cm distal to the elbow crease other sites are discouraged because proximal cuff postioning affects the magnitude of the peak vasodilatory response occlusion time: 5 minutes (shorter inflation attenuates FMD response) cuff inflation to at least 50 mmHg above systolic pressure
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Site selection
brachial artery with a minimum diameter (usually >2 mm). Small arteries are difficult to measure and changes in absolute diameter correspond to big relative changes if repetitive measurements are planned, site has to been replicated -anatomic landmarks should be used.
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Image acquisition
longitudinal images obtained by high-resolution ultrasound (7.5–12 MHz) a clear interface between the near and far arterial wall should be achieved diameter measurements are obtained in end-diastole or averaged over the heart cycle stereotactic adjustable prop-holding is essential to endure image quality recording of the baseline diameter for at least 1 min simultaneous acquisition of pulse-wave Doppler velocity signals for quantification of shear stress (stimulus), if feasible. Insonation angle should be <60°
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Measurement
automated edge-detection should be used reported as maximal percentage change from baseline diameter (most reproducible) report also baseline diameter, absolute change characterization of the hyperemic stimulus (ideally the flow-velocity time integral)
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