Nitrous oxide (N2O) is a potent greenhouse gas with a rising atmospheric concentration largely due to the applications of nitrogen fertilisers globally. Development of mitigating strategies requires accurate estimations of N2O emissions, however, estimates of N2O emissions are uncertain due to its variability in space and time. An area of highest uncertainty is the diurnal variability of soil N2O fluxes due to challenges of highly-frequent measurements. This research aimed to (1) investigate the prevalence of diurnal variability of N2O flux from agricultural soils, (2) evaluate the efficacy of non-diurnal sampling intervals by comparisons with diurnal measurements of N2O flux, and (3) examine the environmental and biological factors driving the diurnal variability of N2O flux. Through the systematic review of published N2O flux data and field- and laboratory-based experiments, this research showed diurnal variability of N2O flux is prevalent in agricultural soils, often exhibiting high diurnal amplitudes (>100%). Afternoon peaking of N2O flux was the most common occurrence (~60% of the time) but its consistency can be impacted by changing field conditions such as interruption by rainfall. The review revealed that a single-daily flux measurements at 10:00 provided the best estimate of the daily mean value of N2O emission (+2%), but with a risk of under- (-29%) or overestimations (+35%). In field mesocosm experiments, diurnal N2O fluxes were collected with a novel automated chamber system. The results of the first trial demonstrated that a single-daily flux measurements could adequately estimate N2O emissions (+7%), whereas once-a-week basis measurements had large associated biases (46±108%). Crucially, these experiments revealed that soil temperature showed little relationship with diurnal N2O flux, whereas evidence of photosynthetic parameters driving diurnal variability of N2O flux were found. The findings in the reductionist laboratory experiment, where fluctuations of soil temperature and moisture were minimised, further suggest PAR-driven plant metabolism is likely a driver of the diurnal variability of N2O flux.