The use of Multi-Voxel Pattern Analysis (MVPA) has increased considerably in recent functional magnetic resonance imaging (fMRI) studies. A crucial step consists in the choice of a method for the estimation of responses. However, a systematic comparison of the different estimation alternatives and their adequacy to predominant experimental design is missing. In the current study we compared three pattern estimation methods: Least-Squares Unitary (LSU), based on run-wise estimation, Least-Squares All (LSA) and Least-Squares Separate (LSS), which rely on trial-wise estimation. We compared the efficiency of these methods in an experiment where sustained activity needed to be isolated from zero-duration events as well as in a block-design approach and in a event-related design. We evaluated the sensitivity of the t-test in comparison with two non-parametric methods based on permutation testing: one proposed in Stelzer et al. (2013), equivalent to performing a permutation in each voxel separately and the Threshold-Free Cluster Enhancement. LSS resulted the most accurate approach to address the large overlap of signal among close events in the event-related designs. We found a larger sensitivity of Stelzer’s method in all settings, especially in the event-related designs, where voxels close to surpass the statistical threshold with the other approaches were now marked as informative regions. Our results provide evidence that LSS is the most accurate approach for unmixing events with different duration and large overlap of signal. This is consistent with previous studies showing that LSS handles large collinearity better than other methods. Moreover, Stelzer’s potentiates this better estimation with its large sensitivity.