To evaluate the time course of EMG frequency changes during a three-minute maximal effort cycling test (3MT) session and to examine which parameter between mean (MNF) and median (MDF) frequency is more suitable for evaluation of changes in neuromuscular function throughout a 3MT. Eighteen recreationally-active men volunteered to participate in this study. Maximum voluntary contraction (MVC) was measured using a dynamometer to determine maximal EMG frequency of the vastus lateralis (VL) of the kicking leg during isometric knee extension. A maximal oxygen consumption test (VO₂peak) on a cycle ergometer was performed to establish the appropriate load profile for the 3MT which was completed after a period of at least 48 hours. MNF, MDF and power output (PO) values were measured at 10-second epochs throughout the duration of the 3MT. Repeated measures analysis of variance was used to compare the changes in EMG frequency, relative to maximal values from the MVC, and change in PO during the testing procedure. MNF, Root Mean Square (RMS), and PO significantly decreased during the 3MT, while MDF did not change significantly. Statistically, EMG frequency and PO decreased at first and remained constant in response to the 3MT, which may be reflective of differing patterns of muscle fiber type fatigue throughout the testing session. Due to decreased variability, changes in neuromuscular function during this protocol may be better evaluated using MNF than MDF.