Poor regulation and maintenance of circulating calcium is of concern in dairy cows, particularly during the peri-parturient period as cows adapt to increased demands of colostrum and milk production. When regulation of circulating calcium fails, hypocalcemia ensues, with repercussions of this disease negatively affecting cow health throughout early lactation. The objective of this study was to characterize physiologic responses of a subclinical hypocalcemic challenge in lactating and nonlactating dairy cows. Using a randomized complete block design, 12 dry, non-pregnant multiparous Holstein cows and 12 early lactation (5–20 DIM) multiparous Holstein cows received either (1) a continuous 24-h intravenous solution of 0.9% saline or (2) 5% ethylene glycol tetraacetic acid (EGTA) in 0.9% saline (n = 6 lactating, n = 6 nonlactating/treatment) with the aim of maintaining blood ionized calcium (iCa) less than 1.0 mM. Blood samples were collected immediately before infusion, hourly during infusion, and 4, 8, 12, 24, 48, and 72 h post-infusion to monitor blood iCa concentrations. Groups were compared using a mixed model ANOVA with time included as a repeated measure. Infusion of EGTA effectively decreased circulating iCa concentrations in both lactating and dry, non-pregnant cows compared with saline infusion (0.90 ± 0.01 mM, 0.84 ± 0.01 mM vs 1.25 ± 0.01 mM, 1.23 ± 0.01 mM; P < 0.0001). Lactating-EGTA infused cows had higher iCa during the infusion period compared with dry, non-pregnant EGTA cows (P = 0.003). Lactating-EGTA cows had higher iCa concentrations than all other treatments (1.36 ± 0.026 mM; P < 0.05) 24 h post-infusion. Additionally, dry, non-pregnant, EGTA cows required less EGTA to maintain subclinical hypocalcemia compared with lactating EGTA cows (P < 0.0001). This data suggests that homeostatic response to perturbation of calcium metabolism differs among early lactation and dry non-pregnant cows. Additionally, this demonstrates the early lactation dairy cow is capable of adapting to calcium challenges more rapidly and has improved feedback mechanisms to maintain circulating calcium concentrations than a dry, non-pregnant cow.