For the HADS test, the subjects filled out the forms 1 month before aromatherapy massage, immediately before the first, fifth and eighth massage and 1 month after massage. HADS was used for determination of long-term effect. Procedure of Aromatherapy Massage The subjects received a 30 min aromatherapy massage twice a week for 4 weeks (eight times in total). Each subject received the same standardized massage performed on the anterior area of the neck and thorax, back, shoulders, arms, hands and upper legs for 30 min by skilled therapists. Methods for Immune Measurements Peripheral blood was drawn 1 month before aromather- apy massage, immediately before the first, fifth and eighth massage, and 1 month after massage. Blood cell counts including numbers and proportions of leukocytes were examined at the Central Laboratory Unit of Kyoto Prefectural University of Medicine. Heparinized blood samples of 2 ml were used to determine the levels of various lymphocyte subsets. Mononuclear cells were isolated by Ficoll-PaqueTM PLUS (Amersham Biosciences AB, Sweden) gradient centrifugation. Lymphocyte subsets were identified by FACS analysis (FACS Caliber, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) using FITC (fluorescein- isothiocyanate)-CD16, PE (Phycoerythrin)-CD8, FITC- CD4 antibodies (Becton, Dickinson and Company), CFS (carboxyfluorescein succinimidylester)-CXCR3 antibodies (DAKO A/S, Glostrup, Denmark) and PE-CCR4 (BD Bioscience Pharmingen, San Jose, CA, USA). Statistical Analysis We examined differences in data between pre- and post- massage sessions using the paired t-test. Temporal changes in the scores of psychologic tests were examined using the repeated measures ANOVA and Bonferroni test. All statistical analyses were conducted using SPSS software (Advanced Models 14.0J). A P-value 5 0.05 was considered significant. Results Anxiolytic and Anti-Depressive Effects We assessed psychologic responses to treatment as changes in anxiety and depression using HADS and STAI (Fig. 1). In HADS, we tested the anxiety level 1 month before aromatherapy massage, immediately before the first, fifth and eighth massage and 1 month eCAM 2009;6(1) 125 after massage. The results showed a significant difference among whole periods, tested by repeated measures ANOVA (P50.01). It was found that there were no significant changes in anxiety or depression in HADS during the waiting control period and the anxiety level gradually reduced over time and that there were significant differences between anxiety scores 1 month before massage and immediately before the eighth massage (P50.05) and between those tested 1 month before and after massage (P50.01, by paired t-test and P50.05, by Bonferroni test). However, there were no significant differences observed in the depression scores, by repeated measures ANOVA. Also, in the total HADS score, there were significant differences among whole periods and between those of 1 month before massage and immediately before the eighth massage P50.01, by paired t-test and P50.05 by Bonferroni test), and between those of 1 month before and after massage (P50.01 by paired t-test and Bonferroni test), as in the anxiety scores. In STAI, trait anxiety was gradually reduced and there were significant differences among whole periods, between the scores tested 1 month before massage and immediately before the first massage (P50.05) and between those tested 1 month before and after the first massage (P50.05). State anxiety scores from STAI were significantly reduced after aromatherapy massage. Namely, there were significant differences among whole periods and between the scores tested 1 month before massage and immediately before the fifth massage and between those tested immediately before and after the first, fifth and eighth massages. However, there was no significant difference in the waiting control period. Changes of Mood State Next, we examined the POMS using the POMS test. The results showed that aggression-hostility and fatigue were gradually reduced, but there were no significant differ- ences in anger-hostility between scores tested 1 month before massage and immediately before the fifth massage and between scores tested 1 month before massage and immediately before the eighth massage. A significant difference was observed in aggression-hostility between scores tested 1 month before and after massage (P50.05). Immunologic Measures There were no significant differences in the number of leukocytes, and neutrophils, lymphocyte, CD4-positive lymphocytes and CD8-positive lymphocytes which are types of leukocytes, in the waiting control period. Leukocytes and lymphocyte increased significantly after the eighth aromatherapy massage session, as compared with those before the eighth massage (Fig. 2).