Blood oxygenation level-dependent (BOLD) functional MRI is a widely employed methodology in experimental and clinical neuroscience, although its nature is not fully understood. To gain insights into BOLD mechanisms and take advantage of the new functional methods, it is of interest to investigate prolonged paradigms of activation suitable for long experimental protocols and to observe any long-term modifications induced by these functional challenges. While different types of sustained stimulation paradigm have been explored in human studies, the BOLD response is typically limited to a few minutes in animal models, due to fatigue, anesthesia effects and physiological instability. In the present study, the rat forepaw was electrically stimulated for 2 h, which resulted in a prolonged and localized cortical BOLD response over that period. The stimulation paradigm, including an inter-stimulus interval (ISI) of 10 s, that is 25% of the total time, was applied at constant or variable frequency over 2 h. The steady-state level of the BOLD response was reached after 15-20 min of stimulation and was maintained until the end of the stimulation. On average, no substantial loss in activated volume was observed at the end of the stimulation, but less variability in the fraction of remaining activated volume and higher steady-state BOLD amplitude were observed when stimulation frequency was varied between 2 and 3 Hz every 5 min. We conclude that the combination of ISI and variable stimulus frequency reproducibly results in robust, prolonged and localized BOLD activation. Copyright © 2015 John Wiley & Sons, Ltd.