Following lower limb injury, some patients are not able to walk at full weight bearing and may require body weight support for ambulation during the early stages of rehabilitation. The aim of the present study was to investigate how various degrees of reduced effective body weight in a Lower Body Positive Pressure Treadmill (LBPPT), affects muscle activation levels during walking. Twelve healthy participants were instructed to walk at 2.5 km/h and 3.6 km/h on a LBPPT that provided a reduced effective body weight equivalent to 100%, 80%, 60%, 40%, and 20% of their individual body mass. Electromyography data were recorded during 20 gait cycles, from seven lower limb muscles, and segmented into a mean envelope by computing root mean square values. A two-way repeated measures ANOVA was used to test for differences in the highest root mean square value obtained, with walking speed and fractional reduction in effective body weight as factors. Significant decreases in EMG amplitude were identified in the following muscles as a result of reduced effective body weight: Vastus Medialis, Vastus Lateralis, Soleus, Gastrocnemius Medial and Lateral head (p ≤ 0.05). For Tibialis Anterior, significant reductions in EMG amplitude were only observed when effective body weight was reduced to 40% or less at a walking speed of 2.5 km/h (p ≤ 0.05). The EMG amplitude for Tibialis Anterior at 3.6 km/h and Biceps Femoris at both speeds remained unaffected at all fractional reductions (p ≥ 0.05). These findings suggests that the muscles of the lower limb respond differently to the body weight support provided by the LBPPT during walking, with the extensor muscles of the knee and ankle displaying decreased muscle activation, and the Tibialis Anterior and Biceps Femoris displaying minimal to no changes in muscle activation.