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Gene expression profiling is one of the most reliable high-throughput phenotyping methods, allowing researchers to quantify the transcript abundance of expressed genes. Because many biotic and abiotic factors influence gene expression, it is recommended to control them as tightly as possible. Here, we show that a 24 h age difference of Drosophila simulans females that were subjected to RNA sequencing (RNA-Seq) five and six days after eclosure resulted in more than 2000 differentially expressed genes. This is twice the number of genes that changed expression during 100 generations of evolution in a novel hot laboratory environment. Importantly, most of the genes differing in expression due to age introduce false positives or negatives if an adaptive gene expression analysis is not controlled for age. Our results indicate that tightly controlled experimental conditions, including precise developmental staging, are needed for reliable gene expression analyses, in particular in an evolutionary framework.