The purpose of this study is to analyze the long-term riders’ subjective responses to a standardized bikefitting method on their bicycles. Eighty-six amateur mountain bikers had their riding posture and bicycle components ergonomically adjusted through a 3D kinematic bikefitting method. Validated subjective scales (Feeling, OMNI, and Numerical Rating Pain Scale) were used to assess their overall riding comfort and fatigue along with localized pain for six body parts. Data were collected just before intervention (baseline or pre), immediately after (or post), and 30, 60, 90, and 120 days after the bikefit session. A Student’s t-test comparing before bikefit and after 120 days showed significant (p < 0.05) reduction in localized pain for all six body parts and riding comfort along with a large effect size effect (d = 1.18) for riding comfort. Although initially reduced, fatigue scores gradually increased over the months, showing a high correlation (r = 0.946) with increased monthly training volume. In conclusion, overall riding discomfort and pain were significantly decreased after a standardized kinematic bikefit session even after 120 days post intervention. However, fatigue scores began to rise after 30 days, showing a high correlation with increasing monthly training volume.