This study was designed to assess the effects of acute exercise on performance of a paired associate learning (PAL) test, an operationalization of hippocampal-dependent associative memory. Participants performed a PAL test and then ran on a treadmill (exercise group, n = 52) or solved Sudoku puzzles (control group, n = 54). Participants returned 2, 5, or 8 hr later to perform a second, different, PAL test. PAL scores for the control group did not change over time. Similarly, scores on tests taken 2 and 5 hr after exercise were not different from baseline or control data. Scores on tests taken 8 hr after exercise, however, fell significantly below baseline (by 8.6%) and control (by 9.8%) scores. These data demonstrate that acute exercise can negatively affect the encoding and retrieval of new information even hours after the exercise bout, which should be a consideration when designing exercise programs to enhance, and not hinder, learning.