In all, 60 Angus×Hereford heifers were ranked by age and BW (210±2 days and 220±2 kg) on day 0, and assigned to: (a) one of three drylot pens (10×14 m pens; 10 heifers/pen) resulting in a stocking density of 14 m2/heifer (HIDENS; n=3), or (b) one of three pastures (25 ha pastures; 10 heifers/pasture), resulting in a stocking density of 25 000 m2/heifer (LOWDENS; n=3). Pastures were harvested for hay before the beginning of this experiment, and negligible forage was available for grazing to LOWDENS heifers during the experiment (days 0 to 182). All heifers received the same limited-fed diet, which averaged (dry matter basis) 4.0 kg/heifer daily of hay and 3.0 kg/heifer daily of a corn-based concentrate. Heifer shrunk BW was recorded after 16 h of feed and water withdrawal on days −3 and 183 for BW gain calculation. On day 0, heifers were fitted with a pedometer behind their right shoulder. Each week, pedometer results were recorded and blood samples were collected for puberty evaluation via plasma progesterone. Plasma samples collected on days 0, 28, 56, 84, 112, 140, 161 and 182 were also analyzed for cortisol concentrations. On days 0, 49, 98, 147 and 182, hair samples were collected from the tail switch for analysis of hair cortisol concentrations. On days 28, 102 and 175, blood samples were collected for whole blood RNA isolation and analysis of heat shock protein (HSP) 70 and HSP72 mRNA expression. Heifers from LOWDENS had more (P<0.01) steps/week compared with HIDENS. No treatment effects were detected (P=0.82) for heifer BW gain. Plasma cortisol concentrations were greater (P⩽0.05) in LOWDENS compared with HIDENS heifers on days 84, 140, 161 and 182 (treatment×day interaction; P<0.01). Hair cortisol concentrations were greater (P<0.01) in HIDENS compared with LOWDENS heifers beginning on day 98 (treatment×day interaction; P<0.01). Heifers from LOWDENS had greater (P=0.04) mean mRNA expression of HSP72, and tended (P=0.09) to have greater mean mRNA expression of HSP70 compared with HIDENS. Heifers from HIDENS experienced delayed puberty attainment and had less (P<0.01) proportion of pubertal heifers on day 182 compared with LOWDENS (treatment×day interaction; P<0.01). In summary, HIDENS altered heifer stress-related and physiological responses, and delayed puberty attainment compared with LOWDENS.