The first aim of this study was to compare an ambulatory five-lead ECG system with the commercially available breast belt measuring devices; Polar S810i and Suunto t6, in terms of R-R interval measures and heart rate variability (HRV) indices. The second aim was to compare different HRV spectral analysis methods. Nineteen young males (aged between 22 and 31 years, median 24 years) underwent simultaneous R-R interval recordings with the three instruments during supine and sitting rest, moderate dynamic, and moderate to vigorous static exercise of the upper and lower limb. For each subject, 17 R-R interval series of 3-min length were extracted from the whole recordings and then analyzed in frequency domain using (1) a fast Fourier transform (FFT), (2) an autoregressive model (AR), (3) a Welch periodogram (WP) and (4) a continuous wavelet transform (CWT). Intra-class correlation coefficients (ICC) and Bland-Altman limits of agreement (LoA) method served as criteria for measurement agreement. Regarding the R-R interval recordings, ICC (lower ICC 95% confidence interval >0.99) as well as LoA (maximum LoA: -15.1 to 14.3 ms for ECG vs. Polar) showed an excellent agreement between all devices. Therefore, the three instruments may be used interchangeably in recording and interpolation of R-R intervals. ICCs for HRV frequency parameters were also high, but in most cases LoA analysis revealed unacceptable discrepancies between the instruments. The agreement among the different frequency transform methods can be taken for granted when analyzing the normalized power in low and high frequency ranges; however, not when analyzing the absolute values.