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 three different pedaling frequency (n = 5): 70, 80, and 90 rpm. From 30 to 50% of the maximal power output (P(max)), in the 90 rpm 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) did not increase significantly from its minimal values in the 70 rpm ( approximately 1.26 ms) and 80 rpm ( approximately 1.20 ms) groups. Then, from 60 to 100% maximal power output (P(max)), workload increase caused a significant approximately 2.8-, approximately 3.3-, and approximately 3.4-fold increase in A(PFC) in the 70, 80, and 90 rpm groups, respectively, with no significant difference between groups. At peak exercise, A(PFC) accounted for approximately 43, approximately 39, and approximately 49% of the total HRV in the 70, 80, and 90 rpm 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.