The aim was to develop a half-udder model for quantifying the local effects of elevated udder temperature on mammary gland (MG) function in dairy cows. Heating pads were used to heat the MG. Four settings were tested and were found to be stable after 45 min at significantly different temperatures (P < 0.001; Warm = 43.4, Low = 45.8, Medium = 48.6, and High = 50.4 ± 0.5°C). Heating pads were then applied to half-udders of individual cows. Two cows were used to test each temperature setting; for each cow, one half-udder served as heated treatment and the other half-udder was the unheated control. During each 4-h test period, cows were monitored every 20 min. There was no evidence of blisters, inflammation, redness, sensitivity to touch or any sign of discomfort. Udder skin temperature increased in a heat-setting dependent manner (P ≤ 0.01). Milk temperature was higher in heated than in unheated halves (P < 0.001; 37.4 vs. 36.9 ± 0.04°C, respectively), but no differences over time or between heat-settings were observed. Next, heat was applied to half-udders of 4 cows for 48 h, and individual halves were milked every 12 h. Udder skin temperature increased in the heated compared with unheated halves (P < 0.001; 38.6 vs 36.4 ± 0.08°C, respectively). Somatic cell counts and milk composition were similar between udder halves throughout the trial. At the 48 h milking, yield was reduced by 0.8 ± 0.2 kg in heated halves compared with unheated halves (P < 0.05). RNA from milk somatic cells collected from each udder-half at 48 h was sequenced, and a total of 151 annotated genes were differentially expressed (P ≤ 0.001 and FDR ≤0.10) between the heated and unheated halves. These genes represented several functional clusters, including cell membrane, signaling, inflammatory and stress response, apoptotic processes, and mammary gland development. Pathways found to be differentially expressed between groups included cytokine-cytokine receptor integration, chemokine and TNF signaling, coagulation and complement cascade, and others. These experiments established a half-udder model for investigating local effects of heat stress on mammary function.