The objectives of this study were (1) to evaluate the variability in spot short-term measurements of CO₂ (QCO₂) CH₄ (QCH₄) emissions and O₂ consumption (QO₂), and (2) estimate heat production (HP) from dairy cows fed diets with different RDP and NSC profiles. Eight multiparous and 4 primiparous lactating Holstein cows were blocked by days in milk and milk yield into replicated 4 × 4 Latin squares. Cows received corn silage, grass-legume haylage, roasted soybean, and (1) corn meal and urea (negative control), (2) corn meal, soybean meal, and rumen-protected Lys-Met (positive control), (3) field peas, corn meal, and soybean meal, or (4) field peas, corn meal, soybean meal, and rumen-protected Lys-Met. Gas measurements were performed using a portable automated gas quantification system (Greenfeed, C-Lock Inc., Rapid City, SD) fitted with sensors for CO₂, CH₄ and O₂. Cows were sampled 3 times daily (about 8 h apart) for 7 d, with sampling points advanced 2 h in the following day to account for diurnal variation of gas flux. Heart rate was monitored for 4 d to calculate QO₂/heartbeat (n = 4 cows). Heat production was calculated according to Kaufmann et al. (2011): [(4.96 + 16.07/respiratory quotient) × QCO₂]. Filtering of gas sampling by variable head position or low visitation to the gas quantification system (n = 2) resulted in lower number of gas flux points than anticipated (316 valid measurements of 1,008 possible). Each gas sampling lasted, on average, 3.7 min. For all treatments, the within animal CV averaged 25.5% (QCH₄), 15.4% (QCO₂), and 22.3% (QO₂) and the between animal CV averaged 7.0% (QCH₄), 4.3% (QCO₂), and 5.8% (QCO₂). There was no difference between treatments for QCH₄ (mean = 373.5 g/d), QCO₂ (mean = 6,293 L/d), QO₂ (mean = 7,307 L/d), HP (mean = 149 MJ/d), QO₂/heartbeat (mean = 66.1 L/heartbeat), and HP/heartbeat (mean = 20.7 MJ/heartbeat). Short-term spot measurements of gas flux can be used to estimate HP in dairy cows. However, sampling rate needs to be optimized, when using the automated gas quantification system, to reduce variability.