Sample size calculation is a crucial step in study design but is not yet fully established for RNA sequencing (RNA-seq) analyses. To evaluate feasibility and provide guidance, we evaluated RNA-seq sample size tools identified from a systematic search. The focus was on whether real pilot data would be needed for reliable results and on identifying tools that would perform well in scenarios with different levels of biological heterogeneity and fold changes (FCs) between conditions. We used simulations based on real data for tool evaluation. In all settings, the six evaluated tools provided widely different answers, which were strongly affected by FC. Although all tools failed for small FCs, some tools can at least be recommended when closely matching pilot data are available and relatively large FCs are anticipated.