The objective of this study was to examine whether stress responsiveness and metabolism differed in cattle of divergent temperament and muscling characteristics when handling and activity were minimised. Hypothalamic-pituitary-adrenal (HPA) axis function, and metabolic indicators were measured in 20 Angus steers (mean ± SD, 464 ± 6.6 kg liveweight, 17 ± 0.1 m of age) with divergent temperament and muscling. Temperament was assessed using two behavioural tests: flight speed (FS; m/s) and crush score (CS; 1-5); and muscling was assessed using a visual muscle score (MS; 1-15). Calm vs. Flighty steers (n = 10/group) had yearling FS of 1.0 vs. 2.0 m/s, (P < 0.001) and MS of 6.5 vs. 6.7 (P = 0.72). Low vs. High muscled steers (n = 10/group) had yearling MS of 4.4 vs. 8.8 (P < 0.001) and yearling FS of 1.4 vs. 1.5 m/s (P = 0.62). The steers were habituated to handling, catheterized, and housed in individual pens. The HPA axis reactivity to exogenous adrenocorticotropin (0.4 and 2 μg/kg BW) and corticotropin releasing hormone (0.02 and 0.08 μg/kg BW) was tested at 4 day intervals for all steers. There were no differences in baseline HPA axis activity due to temperament or muscling (all P ≥ 0.18) and no difference in HPA axis response to challenge (all P ≥ 0.11). Flighty cattle had higher baseline plasma non-esterified fatty acid (NEFA) concentration (P = 0.011), higher post-challenge plasma glucose (P = 0.027) and lactate (P = 0.045) concentrations, and a tendency towards higher post-challenge plasma NEFA concentration (P = 0.06) than Calm cattle. There were no differences in plasma metabolites with muscling, and no interaction with temperament. These findings indicate that although the HPA axis functioned similarly in Calm and Flighty cattle when handling and activity were minimised, the Flighty cattle remained more metabolically responsive.