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The objective was to characterize physiological responses to unmitigated surgical castration in calves of varying ages. Thirty male Holstein calves of three ages [<6 w (6W); 3 m (3M); 6 m (6M); n = 10] underwent a simulated castration treatment (SHAM) followed 24 h later by castration (CAST). For both treatments, heart rate variability, eye temperature, and cortisol were measured over time from treatment to specified end points to capture the acute response period. Interactions between treatment and age ( p = 0.035) and time and age ( p < 0.001) were noted for cortisol. The 6W calves had lower cortisol compared to 6M calves at SHAM and CAST. Cortisol of 6W calves decreased from peak to pre-treatment levels faster than 6M calves. An interaction between time and age was reported in squared differences of inter-beat-intervals (RMSSD; p = 0.02) and high-frequency power (HFP; p = 0.05), whereby both responses decreased in 6W calves during the sampling period which was not seen in 3M and 6M calves. Average eye temperature (AET) differed by age ( p = 0.0018) whereby 6W calves had lower AET than 6M calves ( p = 0.0013) regardless of treatment and time. The findings suggest that responses to unmitigated surgical castration seem to be mediated by the autonomic nervous system in an age-related manner.