Background: The relationship between exercise and lifespan is well documented, but little is known about the effects of specific exercise protocols on modern measures of biological age. Transcriptomic age predictors provide an opportunity to test the effects of high intensity interval training (HIIT) on biological age utilizing whole-genome expression data. Methods: A single-site, single-blinded, randomized controlled clinical trial design was utilized. Thirty sedentary participants (aged 40 to 65) were assigned to either a HIIT group or a no-exercise control group. After collecting baseline measures, HIIT participants performed three 10X1 HIIT sessions per week for 4 weeks. Each session lasted 23 minutes, and total exercise duration was 276 minutes over the course of the 1-month exercise protocol. Transcriptomic age, PSS-10 score, PSQI score, PHQ-9 score, and various measures of body composition were all measured at baseline and again following the conclusion of exercise/control protocols. Results: Transcriptomic age reduction of 3.59 years was observed in the exercise group while a 3.28-year increase was observed in the control group. PHQ-9, PSQI, BMI, body fat mass, and visceral fat measures were all improved in the exercise group. A hypothesis-generation gene expression analysis suggested exercise may modify autophagy, mTOR, AMPK, IP3K, neurotrophin signaling, insulin signaling, and other age-related pathways. Conclusion: A low dose of HIIT can reduce an RNA-based measure of biological age in sedentary males and females between the ages of 40 and 65. Other changes to gene expression were relatively modest, which may indicate a focal effect of exercise on age-related biological processes.