•We studied changes in stride frequency and body weight support on running mechanics.•Participants ran at their preferred stride frequency (SF) and preferred SF ± 10%.•Participants ran at 0% body weight support (BWS), 50%BWS, and 80%BWS.•A change in SF influenced muscle activity during running, regardless of BWS.•A change in BWS influenced muscle activity, PSF, and preferred speed during running. Running with body weight support (BWS) has been used for physical fitness enhancement. Nevertheless, gait mechanics of running with BWS is not fully understood. We investigated influence of stride frequency manipulation on muscle activity during running at various BWS conditions. Nineteen participants (23.8 ± 4.1 years) ran on a lower body positive pressure treadmill at their preferred speed and preferred stride frequency (PSF) for 0%BWS, 50%BWS, and 80%BWS conditions. Preferred speed and PSF were selected for each of the BWS conditions. The stride frequency conditions consisted of running at PSF, PSF+10%, and PSF-10%. Muscle activity from the rectus femoris (RF), biceps femoris (BF), tibialis anterior (TA), and gastrocnemius (GA) were measured. RF and BF during running at the PSF+10% were higher than when running at the PSF, regardless of BWS (P < 0.01). Additionally, RF and TA during running at the PSF-10% were higher than when running at the PSF, regardless of BWS (P < 0.05). Furthermore, RF, TA, GA, and PSF during running decreased with increasing BWS (P < 0.05), although preferred speed increased with increasing BWS (P < 0.001). These observations suggest that manipulating stride frequency by 10% from the PSF during running produces greater RF, BF, and TA than when running at the PSF, regardless of BWS. Furthermore, it was suggested that a change in BWS influences RF, TA, GA, PSF, and preferred speed during running. Such information may be useful to enable the practitioner to refine the use of running with BWS in exercise programs.