Inflammatory signals in early lactation may aid in the allocation of nutrients toward milk production through altered sensitivity of endocrine signals. We sought to measure effects of anti-inflammatory treatment, as well as the impact of reducing nutrient demand by the mammary gland, on endocrine signaling in early lactation. Multiparous Holstein cows were enrolled at calving and randomly assigned to either sodium salicylate (SS; 2 g/L) or control (CTL; molasses carrier) treatment, administered via drinking water for the first 5 d of lactation, and to a milking frequency (MF) of either once (1×) or 3 times daily (3×), in a 2 × 2 factorial design (n = 8–9). Mixed models were used to assess repeated measures over time. Analysis of plasma samples collected daily before feeding on d 1 to 5 showed that insulin levels were decreased with more frequent milking (0.32 vs. 0.24 ± 0.02 ng/mL; P = 0.02), and also decreased over time compared with 1× (interaction P = 0.02). Insulin was also decreased with SS treatment (0.31 vs. 0.25 ± 0.02 ng/mL for CTL and SS, respectively; P = 0.04). Treatment × MF interactions for plasma glucagon (P = 0.05) and resistin (P = 0.05) revealed increases in both hormones only in 3 × cows treated with SS. Glucagon concentration was greater on d 5 for 3 × vs. 1 × cows (35.5 vs. 24.9 ± 2.9 pg/mL; P = 0.01). Circulating TNFα was also increased by frequent milking (56.1 vs. 34.1 ± 5.3 pg/mL; P < 0.01). On d 5 of the study, cows underwent a glucose clamp protocol with a 2-h euglycemic phase followed by a 2-h hyperinsulinemic-euglycemic phase; blood samples were collected in the last 30 min of each phase. During hyperinsulinemia, SS increased resistin compared with CTL (124 vs. 64 ± 19 pg/mL; P = 0.03). Hyperinsulinemia decreased glucagon only in the 3 × cows (MF × phase interaction P = 0.05). These results suggest that greater MF (and negative energy balance) may promote inflammatory signaling and alter adipose and pancreatic sensitivity to endocrine signals in early lactation. Manipulation by SS treatment triggers additional endocrine responses to maintain metabolic homeostasis in this window of time.