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This work studies the frequency behavior of a least-square method to estimate the power spectral density of unevenly sampled signals. When the uneven sampling can be modeled as uniform sampling plus a stationary random deviation, this spectrum results in a periodic repetition of the original continuous time spectrum at the mean Nyquist frequency, with a low-pass effect affecting upper frequency bands that depends on the sampling dispersion. If the dispersion is small compared with the mean sampling period, the estimation at the base band is unbiased with practically no dispersion. When uneven sampling is modeled by a deterministic sinusoidal variation respect to the uniform sampling the obtained results are in agreement with those obtained for small random deviation. This approximation is usually well satisfied in signals like heart rate (HR) series. The theoretically predicted performance has been tested and corroborated with simulated and real HR signals. The Lomb method has been compared with the classical power spectral density (PSD) estimators that include resampling to get uniform sampling. The authors have found that the Lomb method avoids the major problem of classical methods: the low-pass effect of the resampling. Also only frequencies up to the mean Nyquist frequency should be considered (lower than 0.5 Hz if the HR is lower than 60 bpm). It is concluded that for PSD estimation of unevenly sampled signals the Lomb method is more suitable than fast Fourier transform or autoregressive estimate with linear or cubic interpolation. In extreme situations (low-HR or high-frequency components) the Lomb estimate still introduces high-frequency contamination that suggest further studies of superior performance interpolators. In the case of HR signals the authors have also marked the convenience of selecting a stationary heart rate period to carry out a heart rate variability analysis.