Treadmill walking is a popular form of exercise that offers many benefits to its users, such as improvements in cardiovascular health and gait patterns. Few research studies have explored muscle activation of various lower extremity joints at different levels of inclination on a treadmill. Therefore, this study aims to further characterize muscle activation during gait in healthy individuals in response to changes in treadmill inclination at a constant speed. Twenty healthy participants (24.5 ± 4.3 years of age) were recruited for this study. Participants were instructed to walk on a treadmill at six different inclines (0%, 3%, 6%, 9%, 12%, and 15%) while maintaining a constant speed of 3.4 mph. Muscle activation of the tibialis anterior (TA), gastrocnemius (GA), gluteus maximus (GMAX), gluteus medius (GMED), vastus medialis (QUADS), and biceps femoris (HS) were collected using surface EMG. There were slight differences in muscle activation between the muscle groups during the various intervals. However, there were no significant differences between muscle groups. The results revealed that the extensor muscles (GA, HS, and GMAX) of the lower extremity showed trends of longer activation periods with an increase in inclination. This study found that as inclination increases, activation of the extensor muscles of the lower extremity also increases while walking on a treadmill. The findings of this study will serve as a baseline for research to compare populations with known gait impairments, such as individuals with HIV, post-stroke, or the elderly, to better understand EMG analysis leading to gait deviations or abnormalities with neuromuscular activation.