A single high‑intensity interval workout (10 one‑minute bike sprints) quickly raises the level of humanin—a tiny protective peptide made by mitochondria—in both muscle and blood. Repeating the workouts over two weeks doesn’t keep resting humanin higher, but each new session still gives a spike. The rise isn’t because the body makes more humanin mRNA, so it’s likely a rapid, non‑gene‑level response.

Humanin is a small regulatory peptide encoded within the 16S ribosomal RNA gene (<i>MT-RNR2</i>) of the mitochondrial genome that has cellular cyto- and metabolo-protective properties similar to that of aerobic exercise training. Here we investigated whether acute high-intensity interval exercise or short-term high-intensity interval training (HIIT) impacted skeletal muscle and plasma humanin levels. Vastus lateralis muscle biopsies and plasma samples were collected from young healthy untrained men (<i>n</i> = 10, 24.5&#x2009;&#xb1;&#x2009;3.7 yr) before, immediately following, and 4 h following the completion of 10&#x2009;&#xd7;&#x2009;60 s cycle ergometer bouts at V&#x307;o_2peak power output (untrained). Resting and postexercise sampling was also performed after six HIIT sessions (trained) completed over 2 wk. Humanin protein abundance in muscle and plasma were increased following an acute high-intensity exercise bout. HIIT trended (<i>P</i> = 0.063) to lower absolute humanin plasma levels, without effecting the response in muscle or plasma to acute exercise. A similar response in the plasma was observed for the small humanin-like peptide 6 (SHLP6), but not SHLP2, indicating selective regulation of peptides encoded by MT-RNR2 gene. There was a weak positive correlation between muscle and plasma humanin levels, and contraction of isolated mouse EDL muscle increased humanin levels ~4-fold. The increase in muscle humanin levels with acute exercise was not associated with <i>MT-RNR2</i> mRNA or <i>humanin</i> mRNA levels (which decreased following acute exercise). Overall, these results suggest that humanin is an exercise-sensitive mitochondrial peptide and acute exercise-induced humanin responses in muscle are nontranscriptionally regulated and may partially contribute to the observed increase in plasma concentrations.<b>NEW &amp; NOTEWORTHY</b> Small regulatory peptides encoded within the mitochondrial genome (mitochondrial derived peptides) have been shown to have cellular cyto- and metabolo-protective roles that parallel those of exercise. Here we provide evidence that humanin and SHLP6 are exercise-sensitive mitochondrial derived peptides. Studies to determine whether mitochondrial derived peptides play a role in regulating exercise-induced adaptations are warranted.