Across muscle tissue the rhythmical variation in blood flow (flowmotion) is such that at any one time only a proportion of capillaries that supply nutrients to the muscle are perfused. Different factors control blood flow variation, with each contributing factor occurring at a specific frequency; endothelial (0.006-0.02Hz), neurogenic (0.02-0.06Hz), and myogenic (0.06-0.3Hz). A change in the contribution of any of these factors to blood flow regulation can lead to an overall increase or decrease in nutrient delivery to the myocytes, thereby potentially altering the metabolic activity of the muscle tissue. Determining flowmotion within in vivo skeletal muscle is difficult and previously assessment has been performed by invasive studies with implanted Laser Doppler Flowmetry (LDF) probes. LDF only allows flowmotion assessment in a small proportion of skeletal muscle tissue, however this study uses novel real-time contrast enhanced ultrasound (RT-CEU) imaging in the assessment of microvascular blood flow across whole skeletal muscles in vivo. Male Sprague Dawley rats were anaesthetised (i.p sodium pentobarbital, 50mg/100g body weight) and cannulas placed into jugular veins and carotid artery. Anaesthesia was maintained via intravenous infusion (sodium pentobarbital 0.6mg/min/kg body weight) throughout experiment. Phospholipid microbubbles (average 4µm diameter) where infused into the jugular vein and RT-CEU flowmotion assessment in the calf muscle was made with a Philips iU22 ultrasound machine using a L9-3 transducer. QLab (Philips) software was used to analyse regions of interest and wavelet transformation (MATLAB) was used to determine the contribution of endothelial, neurogenic and myogenic induced vasomotion. LDF, oxygen saturation and total haemoglobin measures where simultaneously determined via a probe (CP3-HP, Moor Instruments) placed directly onto the tibilaris muscle and compared to RT-CEU measures. A strong neurogenic contribution to flowmotion was seen in both LDF and RT-CEU measures. Treatment with the adrenergic antagonist phentolamine produced a marked reduction in neurogenic flowmotion contribution while concurrently increasing total blood flow and microvascular perfusion to the skeletal muscle to a similar extent of that seen during muscle contraction. This study shows that RT-CEU is a new minimally invasive technique which may be used to determine in vivo skeletal muscle flowmotion. Application of RT-CEU in animal models and human participants may lead to determination of any changes in flowmotion which occur during alterations in metabolism, such as during insulin infusion. Additionally RT-CEU may lead to greater understanding of the causes of vascular regulation defects which occur during pathological conditions affecting the microvascular such as hypertension, insulin resistance and Type 2 Diabetes. Funded by NHMRC (Australia)