Objectives: To examine the effect of 100 drop punt kicks on isokinetic knee flexor strength and surface electromyographic activity of bicep femoris and medial hamstrings. Design: Randomized control study. Methods: Thirty-six recreational footballers were randomly assigned to kicking or control groups. Dynamometry was conducted immediately before and after the kicking or 10 min of sitting (control). Results: Eccentric strength declined more in the kicking than the control group (p < 0.001; d = 1.60), with greater reductions in eccentric than concentric strength after kicking (p = 0.001; d = 0.92). No significant between group differences in concentric strength change were observed (p = 0.089; d = 0.60). The decline in normalized eccentric hamstring surface electromyographic activity (bicep femoris and medial hamstrings combined) was greater in the kicking than the control group (p < 0.001; d = 1.78), while changes in concentric hamstring surface electromyographic activity did not differ between groups (p = 0.863; d = 0.04). Post-kicking reductions in surface electromyographic activity were greater in eccentric than concentric actions for both bicep femoris (p = 0.008; d = 0.77) and medial hamstrings (p < 0.001; d = 1.11). In contrast, the control group exhibited smaller reductions in eccentric than concentric hamstring surface electromyographic activity for bicep femoris (p = 0.026; d = 0.64) and medial hamstrings (p = 0.032; d = 0.53). Reductions in bicep femoris surface electromyographic activity were correlated with eccentric strength decline (R = 0.645; p = 0.007). Conclusions: Reductions in knee flexor strength and hamstring surface electromyographic activity are largely limited to eccentric contractions and this should be considered when planning training loads in Australian Football.