Characterization of the aerosolized dose emitted from a nebulized system can be determined using CEN (prEN13544-1) methodology and more recently with a Next Generation Impactor (NGI), but evaporative effects can influence the results. We have investigated these characteristics using different flows and cooling with the NGI and compared the results to the standard CEN method using two different nebulizer systems. The NGI was operated using flows of 15 and 30 L min(-1) at room (ROOM) temperature and immediately after cooling at 5 degrees C for 90 min (COLD). Two nebulizer systems, the Sidestream jet nebulizer (SIDE) and the Aeroneb Pro (AERO), were used to nebulize terbutaline sulphate respiratory solution. The CEN method was also used to provide the aerodynamic characteristics of the aerosolized dose from these two nebulizer systems. The mean (SD) mass median aerodynamic diameter (MMAD) using 15COLD, 15ROOM, 30COLD, 30ROOM, and CEN for AERO was 5.0(0.1), 4.1(0.3), 4.4(0.2), 2.0(0.3), and 3.0(1.1) microm, respectively, and 4.2(0.4), 2.6(0.4), 3.5(0.1), 1.7(0.1), and 3.2(0.3) microm for SIDE. The fine particle fraction (FPF), using the NGI, followed the expected trend associated with the corresponding MMAD values, ranging from 48.1 to 70.5% from AERO and 57.3 to 87.8% for SIDE. The mean FPF for AERO and SIDE using the CEN methodology was 72.5 and 63.6%. Overall there was a highly significant difference (p < 0.001) between the different operating conditions for the FPF and MMAD of both nebulizer systems. All methods revealed a significant difference between AERO and SIDE except CEN. Both nebulizer systems were prone to evaporation effects during in vitro testing. Cooling and using a slow flow minimizes evaporation effects with the NGI and should be adopted as the recommended compendial method. The CEN method provides different values to those of the NGI operating conditions and could not differentiate between the two nebulizers.