Experiments were carried out to examine whether innervation zone (IZ) location remains stable at different levels of isometric contraction in the biceps brachii muscle (BB), and to determine how the proximity of the IZ affects common surface electromyography (sEMG) parameters. Twelve subjects performed maximal (MVC) and submaximal voluntary isometric contractions at 10%, 20%, 30%, 40%, 50% and 75% of MVC. sEMG signals were recorded with a 13 rows × 5 columns grid of electrodes from the short head of BB. The IZ shifted in the proximal direction by up to 2.4 cm, depending upon the subject and electrode column. The mean shift of all the columns was 0.6 ± 0.4 cm (10% vs. 100% MVC, P < 0.001). This shift biased the average values of mean frequency (+21.8 ± 9.9 Hz, P < 0.001), root mean square (−0.16 ± 0.15 mV, P < 0.05) and conduction velocity (−1.15 ± 0.93 m/s, P < 0.01) in the channels immediately proximal to the IZ. The shift in IZ could be explained by shortening of the muscle fibers, and thus lengthening of the (distal) tendon due to increasing force. These results underline the importance of individual investigation of IZ locations before the placement of sEMG electrodes, even in isometric contractions.