Knowledge of efficacious dosing respective to exercise type and pain condition is extremely limited in the literature. This study aimed to determine the impact of dose of moderate intensity treadmill walking on experimentally-induced pain in healthy human participants. Forty females were divided into 4 groups: control (no exercise), low dose exercise (3×/wk), moderate dose exercise (5×/wk) or high dose exercise (10×/wk). Over a 7-day period, subjects performed treadmill walking during assigned exercise days. Both qualitative and quantitative measures of pain were measured at baseline, during the trial, and 24 hrs post-final intervention session via sensitivity thresholds to painful thermal and painful pressure stimulation. Significant effects of treatment were found post-intervention for constant pressure pain intensity (p = 0.0016) and pain unpleasantness ratings (p = 0.0014). Post-hoc tests revealed significant differences between control and moderate and control and high dose groups for constant pressure pain intensity (p = 0.0015), (p = 0.0094), respectively and constant pressure pain unpleasantness (p = 0.0040), (p = 0.0040), respectively. Moderate and high dose groups had the greatest reductions in ratings of pain, suggesting that our lowest dose of exercise was not sufficient to reduce pain and that the moderate dose of exercise may be a sufficient starting dose for exercise-based adjuvant pain therapy.