Neck pain is a common musculoskeletal problem often accompanied by reduced exercise-induced hypoalgesia (EIH) or hyperalgesia compared with an asymptomatic population. This study investigated EIH in a healthy population during experimental neck pain. Forty participants were randomized into this double-blinded parallel-group study. On 4 separate test days (day 0, day 2, day 4, and day 15), participants completed the Neck Disability Index and scored neck pain intensity during head movements on a numerical rating scale. At the end of day 0 and day 2, nerve growth factor (NGF) or isotonic saline (control) was injected into the right splenius capitis muscle. Pressure pain thresholds (PPTs) were recorded bilaterally over splenius capitis (neck), temporalis (head), and tibialis anterior (leg) muscles on all days. On day 0, day 4, and day 15, PPTs were recorded before and after a hand-bike exercise. Exercise-induced hypoalgesia was defined as the PPT increase caused by the exercise. Compared with the control group, the NGF group demonstrated higher Neck Disability Index scores at day 2 and day 4 (P < 0.001, η 2> 0.557) and higher numerical rating scale scores (P < 0.03, η 2> 0.09) along with reduced neck PPTs (P < 0.01, d > 0.44) at day 2 (right: 95% confidence interval [CI] [26.0-54.0]; left: 95% CI [6.8-26.9]), day 4 (right: 95% CI [40.5-67.9]; left: 95% CI [6.9-28.2]), and day 15 (right: 95% CI [5.6-37.2]; left: 95% CI [6.9-34.8]). Across days, the EIH effect was reduced at the neck site in the NGF group compared with the control group (P < 0.001, ηp2 = 0.367, 95% CI [-34.5 to -13.7]). At the head and leg sites, the NGF group showed reduced EIH effect compared with the control group (P < 0.05, d > 0.43) on day 4 (head: 95% CI [-61.4 to -22.9]; leg: 95% CI [-154.7 to -72.4]) and day 15 (head: 95% CI [-54.3 to -7.6]; leg: 95% CI [-122.7 to -34.4]). These results indicate that a few days of clinically comparable neck pain and hyperalgesia might have a negative impact on EIH responses and may help explain why some patients with neck pain do not experience immediate positive effects of exercise.