Behavioral guidance systems are commonly used in freshwater ﬁsh conservation. The biological relevance of sound to ﬁsh and recorded responses to human-generated noise supports the viability of the use of acoustics as an effective stimulus in such technologies. Relatively little information exists on the long-term responses and recovery of ﬁsh to repeated acoustic exposures. In a controlled laboratory study, the response and tolerance of Eurasian minnow (Phoxinus phoxinus) shoals to tonal signals (150Hz of 1s pulse duration) differing only in temporal characteristics (“continuous,” “slow,” “intermediate,” or “fast” pulse repetition rate) were investigated. In comparison to independent control groups, ﬁsh increased their mean group swimming speed, decreased inter-individual distance, and became more aligned in response to the onset of all four acoustic treatments. The magnitude of response, and time taken to develop a tolerance to a treatment differed according to pulse repetition rate. Groups were found to have the greatest and longest lasting response to tone sequences tested in this study when they were pulsed at an intermediate rate of 0.2s-1. This study illustrates the importance of understanding the response of ﬁsh to acoustic signals, and will assist toward the development of longer-term effective acoustic guidance systems.