In the present study, we quantitatively described and compared lower extremity neuromuscular patterns during level cycling (LC), 10 and 20% uphill cycling (UC). We hypothesized that both the timing and intensity of activity of selected lower extremity muscles will differ between steep (but not moderate slope) UC condition and LC. Twelve trained mountain bikers performed an experimental test with three different cycling conditions (level, 10% slope and 20% slope) with EMG monitoring of eight lower extremity muscles. Significant changes (p < 0.05) in muscle activation timing during 20% UC compared to LC (15° later onset and 39° earlier offset) were observed in m. rectus femoris (RF). Range of activity during 20% UC compared to LC was also significantly (p < 0.05) modified in m. vastus medialis, m. vastus lateralis (8° and 5° shorter) and m. biceps femoris (BF; 17° longer). Furthermore, a reduction of EMG activity level was observed for RF and m. tibialis anterior (TA) during 20% UC compared to LC (25 and 19%; p < 0.05), while the opposite effect was observed for m. gluteus maximus (GM; 12%; p < 0.05). Peak cross-correlation coefficients in all cycling conditions for all muscles were high (all coefficients ≥ 0.83). We have shown that altered body orientation during steep, but not moderate, slope UC significantly modified the timing and intensity of several lower extremity muscles, the most affected being those that cross the hip joint and TA. The observed modifications in neuromuscular patterns during 20% UC could have a significant effect on lower extremity joint kinetics and cycling efficiency.