Different sampling times for greenhouse gas measurements have been proposed in many incubation studies. Little is known about effects of closure time on denitrification and nitrification rates from incubation experiments. The objectives of this study were to analyse greenhouse gas (carbon dioxide, methane and nitrous oxide) production from different soils with different times of lid closure and to assess effects of different activation times (defined as additional pre-incubation periods before incubation experiments) on gas emissions from soils. Forty grams of air-dried soil samples (depth 0–10 cm) were incubated in 125-mL jars at 25°C with the addition of glucose and nitrate. The first experiment measured greenhouse gas fluxes at different lid-closure times (40, 80, 120 and 1440 min). The second experiment assessed the effects of different durations of soil activation (0.7, 1.3, 2 and 24 h) on gas emissions. Both were conducted with a completely randomised design, with three replicates per treatment. Our findings showed closure time <1 h or >2 h may cause an underestimate of greenhouse gas emissions. Lengthening activation times resulted in different emission rates consistent with soil characteristics. To measure gas fluxes based on linear regression would require four or five sampling points and sampling at a 20-min interval over a maximum period of 80 min for estimating gas fluxes from soil. Because pre-incubation time is critical and a driving factor in the measurement of soil-induced gas emissions, a standardised procedure to quantify gas fluxes is needed for application to other soils.