Voxel Based Morphometry (VBM) studies typically involve a comparison between groups of individuals; this approach however does not allow inferences to be made at the level of the individual. In recent years, an increasing number of research groups have attempted to overcome this issue by performing single case studies, which involve the comparison between a single subject and a control group. However, the interpretation of the results is problematic; for instance, any significant difference might be driven by individual variability in neuroanatomy rather than the neuropathology of the disease under investigation, or might represent a false positive due to the data being sampled from non-normally distributed populations. The aim of the present investigation was to empirically estimate the likelihood of detecting significant differences in gray matter volume in individuals free from neurological or psychiatric diagnosis. We compared a total of 200 single subjects against a group of 16 controls matched for age and gender, using two independent datasets from the Neuroimaging Informatics Tools and Resources Clearinghouse. We report that the chance of detecting a significant difference in a disease-free individual is much higher than previously expected; for instance, using a standard voxel-wise threshold of p<0.05 (corrected) and an extent threshold of 10 voxels, the likelihood of a single subject showing at least one significant difference is as high as 93.5% for increases and 71% for decreases. We also report that the chance of detecting significant differences was greatest in frontal and temporal cortices and lowest in subcortical regions. The chance of detecting significant differences was inversely related to the degree of smoothing applied to the data, and was higher for unmodulated than modulated data. These results were replicated in the two independent datasets. By providing an empirical estimation of the number of significant increases and decreases to be expected in each cortical and subcortical region in disease-free individuals, the present investigation could inform the interpretation of future single case VBM studies. ► Single subject VBM shows high false positive rates in the healthy population. ► Differences in single subjects are more likely to reflect increases than decreases. ► Differences are mainly located in frontal and temporal areas of the neocortex. ► Single subject VBM studies of patients should be interpreted with caution.