Lifelong exercise is associated with regulation of skeletal mass and function, reductions in frailty, and successful aging. Yet, the influence of exercise on myostatin and myostatin-interacting factors is relatively under examined in older males. Therefore, we investigated whether serum total myostatin, free myostatin, follistatin, and growth and differentiation factor 11 (GDF11) were altered following high-intensity interval training (HIIT) in a group of 13 lifelong sedentary (SED; 64  years) and 11 lifelong exercising (LEX; 62  years) older males. SED follistatin was moderately greater than LEX pre-HIIT (Cohen's d = 0.66), and was largely greater post-HIIT (Cohen's d = 1.22). The HIIT-induced increase in follistatin was large in SED (Cohen's d = 0.82) and absent in LEX (Cohen's d = 0.03). GDF11 was higher in LEX pre-HIIT (Cohen's d = 0.49) and post-HIIT (Cohen's d = 0.63) compared to SED. HIIT resulted in no change to GDF11 in LEX or SED (Cohen's d = 0.00–0.03). Peak power output and GDF11 were correlated (r = 0.603), independent of grouping. Differences in GDF11 with lifelong exercise training, paired with the correlation between GDF11 and peak power output, suggested that GDF11 may be a relevant myostatin-interacting peptide to successful aging in humans, and strategies to maintain this need to be further explored.