The use of arterial spin labeling to measure cerebral blood flow (CBF) after acute exercise has not been reported. The aims of this study were to examine: (1) the optimal inversion time to detect changes in CBF after acute exercise and (2) if acute exercise alters CBF in the motor cortex at rest or during finger-tapping. Subjects (n=5) performed 30 min of moderate intensity exercise on an electronically braked cycle ergometer (perceived exertion 'somewhat hard'). Before and after exercise, relative CBF was measured using multiple inversion time (TI) pulsed arterial spin labeling (PASL). Two multiple TI runs were obtained at rest and during 4 Hz finger-tapping. Four inversion times (675, 975, 1275, and 1,575 ms) were acquired per run, with 20 interleaved pairs of tag and control images per inversion time (320 s run). The results indicated that global CBF increased approximately 20% following exercise, with significant differences observed at an inversion time of 1,575 ms (p<.05). Finger-tapping induced CBF in the motor cortex significantly increased from before to after exercise at TI=1,575 ms (p<.01). These findings suggest changes in human cerebral blood flow that result from acute moderate intensity exercise can be detected afterwards using PASL at 3T with an inversion time of 1,575 ms. The effect of prior acute exercise to increase motor cortex CBF during the performance of a motor task suggests future use of indices of functional activation should account for exercise-induced changes in cardio-pulmonary physiology and CBF. Copyright (c) 2010 Elsevier B.V. All rights reserved.