BackgroundProgressive loading of the lower limb muscles during running on a positive pressure or reduced gravity (Alter-G™) treadmill is suggested as a rehabilitation strategy after muscle and tendon injury but the influence of running up or downhill and at higher speeds is not known, nor are the interaction effects of speed, inclination, and indicated bodyweight. Research questionWhat are the lower limb EMG activation levels and cadence when running up and downhill in normal and reduced gravity? Methods10 recreationally active male athletes ran on a positive-pressure Alter-G™ treadmill at: 3 indicated bodyweights (60 %, 80 %, and 100 %); 5 speeds (12, 15, 18, 21, and 24 km/h); for incline, decline, and flat conditions (-15 %, -10 %, -5%, 0%, 5%, 10 %, and 15 %); while monitoring the surface EMG of 11 leg muscles as well as cadence (strides per minute). Results and significanceLinear mixed models showed significant effect of running speed, inclination, and indicated bodyweight, with interaction effects observed. Increasing running speed was associated with the largest change in activity, with smaller effects for increasing bodyweight and inclination. Downhill running was associated with reduced activity in all muscle groups, and more tightly clustered activity patterns independent of speed. Substantial variation in sEMG activity occurred in the flat and uphill conditions. Subject responses were quite variable for sEMG, less so for cadence. For the conditions examined, increasing running speed induced the largest changes in EMG of all muscles examined with smaller changes seen for manipulations of inclination and bodyweight.