Objectives: The purpose of this study was to investigate annual variations in indoor environmental chemical, fungal and dust mite allergen levels, with regards to variations in sick house syndrome (SHS) symptoms over a three-year period. Methods: Detached houses were randomly selected from a building plan approval application, and a questionnaire survey was conducted in 2003 in Sapporo, Japan. Indoor environmental measurements and a self-administered questionnaires survey were conducted on the selected houses in 2004, 2005 and 2006. The same protocol was used for the three-year period to measure the levels of chemicals, fungi and dust mite allergens. A personal questionnaire to assess SHS was distributed to all inhabitants of the dwellings along with one questionnaire to assess housing characteristics. Results: In 2004, 2005 and 2006, the owners of 104, 64 and 41 houses, respectively, agreed to participate in this study. Forty-one houses and the 127 inhabitants who participated in this three-year survey period were included in the analysis to evaluate the associations between differences in environmental measurements and SHS. The levels of formaldehyde, acetone, toluene, Alternaria and Cladosporium tended to decrease, whereas those of limonene and Aspergillus tended to increase over the three-year period. Increasing levels of Cryptococcus and the dampness index in individual houses correlated with increasing SHS symptom scores in the inhabitants after mutual adjustment. Conclusions: Although the average levels of chemicals and fungi were relatively low, the results show the relationship between annual variations in indoor environmental measurements and variations in SHS symptom scores.