Multivariate image analysis has shown potential for classification between Alzheimer's disease (AD) patients and healthy controls with a high-diagnostic performance. As image analysis of positron emission tomography (PET) and single photon emission computed tomography (SPECT) data critically depends on appropriate data preprocessing, the focus of this work is to investigate the impact of data preprocessing on the outcome of the analysis, and to identify an optimal data preprocessing method. In this work, technetium-99methylcysteinatedimer ( 99m Tc-ECD) SPECT data sets of 28 AD patients and 28 asymptomatic controls were used for the analysis. For a series of different data preprocessing methods, which includes methods for spatial normalization, smoothing, and intensity normalization, multivariate image analysis based on principal component analysis (PCA) and Fisher discriminant analysis (FDA) was applied. Bootstrap resampling was used to investigate the robustness of the analysis and the classification accuracy, depending on the data preprocessing method. Depending on the combination of preprocessing methods, significant differences regarding the classification accuracy were observed. For 99m Tc-ECD SPECT data, the optimal data preprocessing method in terms of robustness and classification accuracy is based on affine registration, smoothing with a Gaussian of 12 mm full width half maximum, and intensity normalization based on the 25% brightest voxels within the whole-brain region.