Background Exercise elicits a range of adaptive responses in skeletal muscle, which include changes in mRNA expression. To better understand the health benefits of exercise training, it is important to investigate the underlying molecular mechanisms of skeletal muscle adaptation to exercise. However, most studies have assessed the molecular events at only a few time-points within a short time frame post-exercise, and the variations of gene expression kinetics have not been addressed systematically. Methods We assessed the mRNA expression of 23 gene isoforms implicated in the adaptive response to exercise at six time-points (0, 3, 9, 24, 48, and 72 h post exercise) over a 3-day period following a single session of high-intensity interval exercise. Results The temporal patterns of target gene expression were highly variable and the expression of mRNA transcripts detected was largely dependent on the timing of muscle sampling. The largest fold change in mRNA expression of each tested target gene was observed between 3 and 72 h post-exercise. Discussion and Conclusions Our findings highlight an important gap in knowledge regarding the molecular response to exercise, where the use of limited time-points within a short period post-exercise has led to an incomplete understanding of the molecular response to exercise. Muscle sampling timing for individual studies needs to be carefully chosen based on existing literature and preliminary analysis of the molecular targets of interest. We propose that a comprehensive time-course analysis on the exercise-induced transcriptional response in humans will significantly benefit the field of exercise molecular biology.