Functional adaptation of bone to cyclic fatigue involves a complex physiological response that is targeted to sites of microdamage. The mechanisms that regulate this process are not understood, although lacunocanalicular interstitial fluid flow is likely important. We investigated the effect of a single period of cyclic fatigue on bone blood flow and interstitial fluid flow. The ulnae of 69 rats were subjected to cyclic fatigue unilaterally using an initial peak strain of -6000 muepsilon until 40% loss of stiffness developed. Groups of rats (n=23 per group) were euthanized immediately after loading, at 5 days, and at 14 days. The contralateral ulna served as a treatment control, and a baseline control group (n=23) that was not loaded was also included. After euthanasia, localization of intravascular gold microspheres within the ulna (n=7 rats/group) and tissue distribution of procion red tracer were quantified (n=8 rats/group). Microcracking, modeling, and remodeling (Cr.S.Dn, microm/mm(2), Ne.Wo.B.T.Ar, mm(2), and Rs.N/T.Ar, #/mm(2) respectively) were also quantified histologically (n=8 rats/group). Cyclic fatigue loading induced hyperemia of the loaded ulna, which peaked at 5 days after loading. There was an associated overall decrease in procion tracer uptake in both the loaded and contralateral control ulnae. Tracer uptake was also decreased in the periosteal region, when compared with the endosteal region of the cortex. Pooling of tracer was seen in microdamaged bone typically adjacent to an intracortical stress fracture at all time points after fatigue loading; in adjacent bone tracer uptake was decreased. New bone formation was similar at 5 days and at 14 days, whereas formation of resorption spaces was increased at 14 days. These data suggest that a short period of cyclic fatigue induces bone hyperemia and associated decreased lacunocanalicular interstitial fluid flow, which persists over the time period in which osteoclasts are recruited to sites of microdamage for targeted remodeling. Matrix damage and development of stress fracture also interfere with normal centrifugal fluid flow through the cortex. Changes in interstitial fluid flow in the contralateral ulna suggest that functional adaptation to unilateral fatigue loading may include a more generalized neurovascular response.