Smoking machines were first developed to generate smoke from tobacco cigarettes for the purpose of comparing cigarette tar and nicotine yields under consistent conditions. The International Organization for Standardization (ISO) specifies the atmosphere for the conditioning and testing of tobacco products in reference document ISO 3402:1999E. Conditioning is typically conducted for 48 hours and requires an atmosphere of 22 ±1 °C and 60±3% relative humidity (RH). The testing atmosphere requires 22 ±2 °C and 60±5% RH. No standardized environmental conditions or smoking regimes exist for e-cigarette aerosol collection. Therefore, the purpose of this work was to evaluate the effect of laboratory environmental conditions on the collection of e-cigarette aerosols using a consistent puffing regime. While temperature can typically be controlled in most laboratories, RH cannot. Therefore, RH was the primary focus of this investigation. Commercial e-cigarettes were puffed using a square wave profile for four seconds, 55 cc puff volume, and 30 second puff interval on a 20 port linear smoking machine. Twenty puffs were collected on conditioned Cambridge filter pads (CFP) and the aerosol mass (AM) collected was evaluated for total mass and concentration of nicotine, menthol, propylene glycol, glycerin, and water using gas chromatography with flame ionization and thermal conductivity detectors. Aerosol collection was conducted at 22 ±2 °C with a %RH of 40, 60, and 80. Differences in analyte concentrations at the various RHs will be discussed.