Treadmill-based gait analysis is widely used to investigate walking pathologies and quantify treatment effects on locomotion. Differential sensorimotor conditions during overground vs. treadmill walking necessitate initial familiarization to treadmill walking. Currently, there is no standardized treadmill acclimatization protocol and insufficient familiarization potentially confounds analyses. We monitored initial adaptations to treadmill walking in 40 healthy adults. Twenty-six walking parameters were assessed over 10 minutes with marker-based kinematic analysis and acclimatization profiles were generated. While 16 walking parameters demonstrated initial acclimatization followed by plateau performance, ten parameters remained stable. Distal lower limb control including ankle range of motion, toe trajectory and foot clearance underwent substantial adaptations. Moreover, intralimb coordination and gait variability also demonstrated acclimatization, while measures of symmetry and interlimb coordination did not. All parameters exhibiting a plateau after acclimatization did so within 6-7 minutes (425 strides). Older participants and those naïve to treadmill walking showed adaptations with higher amplitudes but over similar timescales. Our results suggest a minimum of 6 minutes treadmill acclimatization is required to reach a stable performance, and that this should suffice for both older and naïve healthy adults. The presented data aids in optimizing treadmill-based gait analysis and contributes to improving locomotor assessments in research and clinical settings.