β-(1,3)-glucan exposure from household dust has been shown to be associated with respiratory symptoms and thus is increasingly being measured in epidemiological studies. Various factors are known to influence its measurement; however, no studies have assessed the effects of sample extract freeze-thawing on β-(1,3)-glucan. The aim of this study was to assess the effects of repeated freeze-thawing of household dust extracts on levels of β-(1,3)-glucan. Forty random household dust samples were extracted with 0.3 M NaOH and aliquots of extracts stored at −20°C were subjected to one, two, and three freeze-thaw cycles. They were analyzed for β-(1,3)-glucan by the Limulus amoebocyte assay (LAL) and results compared to freshly extracted samples (paired Pearson's t-test on logged values). Initial freezing of house dust extracts results in a significant decline in β-(1,3)-glucan. However, repeated freeze/thawing (up to three times) does not results in any further decline in β-(1,3)-glucan levels.