The amplitude of low-frequency fluctuation (ALFF) measures resting-state functional magnetic resonance imaging (RS-fMRI) signal of each voxel. However, the unit of blood oxygenation level-dependent (BOLD) signal is arbitrary and hence ALFF is sensitive to the scale of raw signal. A well-accepted standardization procedure is to divide each voxel's ALFF by the global mean ALFF, named mALFF. Although fractional ALFF (fALFF), a ratio of the ALFF to the total amplitude within the full frequency band, offers possible solution of the standardization, it actually mixes with the fluctuation power within the full frequency band and thus cannot reveal the true amplitude characteristics of a given frequency band. The current study borrowed the percent signal change in task fMRI studies and proposed percent amplitude of fluctuation (PerAF) for RS-fMRI. We firstly applied PerAF and mPerAF (i.e., divided by global mean PerAF) to eyes open (EO) vs. eyes closed (EC) RS-fMRI data. PerAF and mPerAF yielded prominently difference between EO and EC, being well consistent with previous studies. We secondly performed test-retest reliability analysis and found that (PerAF � mPerAF � mALFF) > (fALFF � mfALFF). Head motion regression (Friston-24) increased the reliability of PerAF, but decreased all other metrics (e.g. mPerAF, mALFF, fALFF, and mfALFF). The above results suggest that mPerAF is a valid, more reliable, more straightforward, and hence a promising metric for voxel-level RS-fMRI studies. Future study could use both PerAF and mPerAF metrics. For prompting future application of PerAF, we implemented PerAF in a new version of REST package named RESTplus. OPEN ACCESS Citation: Jia X-Z, Sun J-W, Ji G-J, Liao W, Lv Y-T, Wang J, et al. (2020) Percent amplitude of fluctuation: A simple measure for resting-state fMRI signal at single voxel level. PLoS ONE 15(1): e0227021. https://doi.org/10.synchronization of the BOLD signals between any pair of brain regions, FC analysis cannot directly measure the regional spontaneous brain activity of a specific voxel per se. Another widely used RS-fMRI measurement, ALFF [2], can be adopted on this purpose, as it provides direct characterization of the spontaneous brain activity at each voxel. However, since BOLD signal has arbitrary units, ALFF is sensitive to the scale of the raw BOLD signal and cannot be directly applied to subsequent group-level statistical analysis.An approach to deal with such a scaling effect is to normalize the raw amplitude value by the global mean amplitude, i.e., the averaged amplitude value across all voxels in the brain [2][3][4]. However, such manipulation assumes that the BOLD signal of all voxels has the same scaling value. In fact, many factors affect the scaling value, including spatial inhomogeneity, brain tissue composition, and brain lesions. Standardization at single voxel level may be helpful to reduce such effects.Another voxel-level metric, namely fractional ALFF (fALFF), had been proposed to normalize ALFF [5]. The fALFF is a ratio of the ALFF within a spec...