It is well known that strenuous eccentric exercise may result in muscle damage. We proposed that vigorous eccentric exercise (EE) would impair myoelectric activity of the biceps brachii. This study utilised a 7-day prospective time-series design. Ten healthy males performed a session of 70 maximal EE elbow flexion contractions. Analysis of surface electromyography activity (sEMG) was performed on the signals recorded during isometric contractions at 50% (IC50) and 80% (IC80) of maximum voluntary isometric torque (MVT), deriving RMS and MDF as sEMG parameters. Linear regression of the RMS and MDF time-series (20-s sustained IC50 and IC80) was used to extract intercepts and slopes of these signals on each day. Plasma creatine kinase activity (CK), MVT, arm circumference, subjective perception of soreness and elbow joint range of motion were also measured to assess effectiveness of EE to evoke muscle damage. CK increased over resting values until day 5 after EE, and remained significantly (p < 0.05) elevated even on day 7. MVT had decreased to 45% of its initial value by day 2 after EE, and remained significantly depressed for the following 6 days. In addition, muscle soreness and arm circumference increased, and range of motion decreased after EE. A significant shift of MDF intercept towards lower frequencies at both IC50 and IC80 was observed after EE in the exercised arm, and these values gradually recovered within the next 3 days during IC50. Although there were some changes in RMS values, these alterations were persistent in both control and exercised arms, and did not follow a consistent pattern. In conclusion, a prolonged reduction in MDF intercept was observed after EE, but this was not closely time-associated with the biochemical, anthropometric or functional markers of muscle damage. Compared to RMS, MDF was a more consistent measure to reflect changes in sEMG.