12 pages, Knowledge of how different management strategies affect gas production from livestock buildings can be helpful for emission predicting purposes and for defining mitigation strategies. The objective of this study was to statistically assess whether and how measured concentrations of ammonia (NH3), methane (CH4) and carbon dioxide (CO2) were influenced by milking frequency. Concentrations of gases were measured continuously by using infrared photoacoustic spectroscopy in the breeding environment of an open dairy barn located in Sicily in hot climate conditions. Data were acquired by specific in-field experiments carried out in 2016 and 2018, when milking sessions occurred twice a day (2MSs) and three times a day (3MSs), respectively. The number of the milking cows was 64 in both 2MSs and 3MSs. The results showed that concentrations of NH3, CH4 and CO2 were statistically influenced by the number of milking sessions. From 2MSs to 3MSs, NH3 concentrations were enhanced (p < 0.001) due to the higher cow’s activity. Conversely, gas concentrations of CH4 and CO2 were lower for 3MSs compared to those for 2MSs due to the effect of the different feeding frequency. Overall, the milking frequency influenced barn management and cow behaviour by modifying the level of gas concentrations in the barn environment.