Contrast-enhanced ultrasound (CEU) has been used to measure muscle microvascular perfusion in vivo in response to exercise and insulin. In the present study we address whether CEU measurement of capillary volume is influenced by bulk flow and if measured capillary filling rate allows discrimination of different flow pattern changes within muscle. Three in vitro models were used: (i) bulk flow rate was varied within a single length of capillary tubing; (ii) at constant bulk flow, capillary volume was increased 3-fold by joining lengths of capillary in series, and compared to a single length; and (iii) at constant bulk flow, capillary volume was increased by sharing flow between a number of lengths of identical capillaries in parallel. The contrast medium for CEU was gas-filled albumin microbubbles. Pulsing interval (time) versus acoustic-intensity curves were constructed and from these, capillary volume and capillary filling rate were calculated. CEU estimates of capillary volume were not affected by changes in bulk flow. Furthermore, as CEU estimates of capillary volume increased, measures of capillary filling rate decreased, regardless of whether capillaries were connected in series or parallel. Therefore, CEU can detect a change in filling rate of the microvascular volume under measurement, but it can not be used to discriminate between different flow patterns within muscle that might account for capillary recruitment in vivo.