Background: In a previous clinical study, levels of biomarkers of exposure (BoEs) for specific toxicants were significantly reduced in smokers who switched from conventional cigarettes to reduced toxicant prototype (RTP) cigarettes. Very little is known about the biological variability of tobacco smoke BoEs within individuals and sub-groups, and the descriptive group-comparison statistics might not be sufficient to understand such changes. Therefore, we assessed how different statistical methods could be used to interpret changes in urine BoE levels at the individual level. Methods: We used non-parametric statistical reference limits, the empirical rule and reference change values (RCVs) to assess changes in levels of BoEs related to four toxicants in cigarettes smoke. Current smokers [of 6 mg and 1 mg International Organization for Standardization (ISO) tar yields] were allocated to switching to RTP groups or non-switching control groups within their respective tar bands. There were two 6 mg tar study groups, with a non-switching group (CC6, n = 46) and a group switching to an RTP containing tobacco-substitute sheet and modified filter (TSS6, n = 49); and three 1 mg tar smoker groups, with one non-switching (CC1, n = 42), a group switching to an RTP containing tobacco-substitute sheet and modified filter (TSS1, n = 44) and one switching to an RTP containing an enzyme-treated tobacco and modified filter (BT1, n = 47). Results: Assessment of the direction of change showed that up to the 100% of subjects experienced a decrease in levels of some BoEs. Between 49% and 64% of subjects in the switching groups were classified as having decreased levels of 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) by the non-parametric criterion, whereas only 2%-6% had reduced levels of N-nitrosoanatabine (NAT). Of non-switchers, in 7%-14% of those smoking 1 mg ISO tar yield cigarettes increases were classified across