Background: Exercise promotes numerous phenotypic adaptations in skeletal muscle that contribute to improved function and metabolic capacity. An emerging body of evidence suggests that skeletal muscle also releases a myriad of factors during exercise, termed “myokines”. The purpose of this study was to examine the effects of high-intensity interval training (HIIT) on the acute regulation of the mRNA expression of several myokines, including the prototypical myokine interleukin-6 (IL-6), and recently identified myokines fibronectin type III domain-containing protein 5 (FNDC5) (irisin) and meteorin-like protein (METRNL). Methods: Both before and after a 20-day period of twice-daily high-volume HIIT, 9 healthy males (20.5 ± 1.5 years performed a standardized bout of high-intensity interval exercise (HIIE; 5 × 4 min at ~80% pretraining peak power output) with skeletal muscle biopsy samples (vastus lateralis) obtained at rest, immediately following exercise, and at 3 h recovery. Results: Before training, a single bout of HIIE increased IL-6 (p