Background Ankle-foot orthoses can be prescribed to improve gait in children with cerebral palsy. Before evaluating the effects of ankle-foot orthoses on gait, a period to adapt or acclimatize is usually applied. It is however unknown whether an acclimatization period is actually needed to reliably evaluate the effect of a new orthosis on gait. This study aimed to investigate whether specific gait parameters in children with cerebral palsy would change within an acclimatization period after being provided with new ankle-foot orthoses. Methods Ten children with cerebral palsy, walking with excessive knee flexion in midstance (8 boys; mean (SD) 10.2 (1.9) years; Gross Motor Function Classification System levels I-II) were provided with ventral shell ankle-foot orthoses. The orthoses were worn in combination with the child's own shoes and tuned, based on ground reaction force alignment with respect to the lower limb joints. Directly after tuning (T0) and four weeks later (T1), 3D-gait analysis was performed using an optoelectronic motion capture system and a force plate. From this assessment, ten spatiotemporal, kinematic and kinetic gait parameters were derived for the most affected leg. Differences in parameters between T0 and T1 were analyzed using paired t-tests or Wilcoxon signed rank tests (P < 0.05). Findings Over the course of four weeks, no significant differences (P > 0.080) were observed for any investigated parameter. Interpretation These results imply that the biomechanical effect of ventral shell ankle-foot orthoses on gait in independent walking children with cerebral palsy is immediately apparent, i.e., there is no further change after acclimatization.