Understanding the consequences and ecological relevance of muscle fatigue is important to guide the development of strategies to preserve independence. However, few studies have examined walking-related fatigue and the effects on walking instability. Our purpose was to investigate the effects of prolonged walking on leg muscle activity and vulnerability to balance perturbations. Eighteen healthy young adults completed a 30-min walking trial at their preferred walking speed while leg muscle activities were recorded. Before and after the 30-min walk, participants responded to five 5% body weight lateral force perturbations. Time-frequency analysis with wavelet transformation and principal component analyses assessed neuromuscular adaptations of muscles to prolonged walking. Following prolonged walking, we observed a time-dependent increase in EMG intensities at slower frequencies for the soleus and tibialis anterior and a decrease in mean amplitudes for the soleus, lateral gastrocnemius, and semitendinosus. Mean mediolateral CoM displacement following perturbations averaged 21% larger after the 30-min walk. Our results suggest that walking for 30 min at a comfortable speed elicits complex neuromuscular adaptations indicative of local muscle fatigue and an increased vulnerability to walking balance perturbations. These findings could inform fatigue monitoring systems or walking assistive devices aimed at reducing walking-related fatigue and maintaining independent mobility.