In order to test the hypothesis that cycling exercise modulates heart rate variability (HRV), we applied a Short-Time Fourier Transform on the electrocardiogram of subjects performing a maximal graded cycling test. A pedaling frequency component (PFC) in HRV was continuously observed over the time course of the exercise test and extracted from R-R interval series obtained from 15 healthy subjects with a heterogeneous physical fitness, exercising at 3 different pedaling frequency (n = 5): 70, 80, and 90 rev.min^-1. From 30% to 50% of the maximal power output (P_MAX), in the 90 rev.min^-1 group, spectral aliasing caused PFC to overlap with the respiratory sinus arrhythmia (RSA) band, significantly overestimating the PFC amplitude (A_PFC). In the meantime, A_PFC didn't increase significantly from its minimal values in the 70 (1.26 ms) and 80 rev.min^-1 (1.20 ms) groups. Then, from 60% to 100% P_MAX, workload increase caused a significant 2.8, 3.3 and 3.4 fold increase in A_PFC in the 70, 80, and 90 rev.min^-1 groups respectively, with no significant difference between groups. At peak exercise, A_PFC accounted for 43%, 39% and 49% of the total HRV in the 70, 80, and 90 rev.min^-1 groups respectively. Our findings indicate that cycling continuously modulates the cardiac chronotropic response to exercise, inducing a new component in HRV, and that workload increase during intense exercise further accentuates this cardiolocomotor coupling. Moreover, because PFC and RSA overlapped at low workloads, methodological care should be taken in future studies aiming to quantify RSA as an index of parasympathetic activity.