This pilot study investigated the differences of heart rate variability (HRV) indices between two opposite emotion states: happiness and sadness, to reveal the differences of autonomic nervous system activity under different emotional states. Forty-eight healthy volunteers were enrolled for this study. Electrocardiography (ECG) signals were recorded under both emotion states with a random measurement order (first happiness emotion measurement then sadness or reverse). RR interval (RRI) time series were extracted from ECGs and multiple HRV indices, including time-domain (MEAN, SDNN, RMSSD and PNN50), frequency-domain (LFn, HFn and LF/HF) and nonlinear indices (SampEn and FuzzyMEn) were calculated. In addition, the effects of heart rate (HR) and mean artery pressure (MAP) on the aforementioned HRV indices were analyzed for both emotion states. The results showed that experimental order had no significant effect on all HRV indices from both happiness and sadness emotions (all P > 0.05). The key result was that among all nine HRV indices, six indices were identified having significant differences between happiness and sadness emotion states: MEAN (P = 0.028), SDNN (P = 0.002), three frequency-domain indices (all P <0.0001) and FuzzyMEn (P = 0.047), whereas RMSSD, PNN50 and SampEn had no significant differences between the two emotion states. All indices, except for SampEn, had significant positive correlations (all P <0.01) for the two emotion states. Four time-domain indices decreased with the increase of HR (all P <0.01), while frequency-domain and nonlinear indices demonstrated no HR-related changes for each emotional state. In addition, all indices (time-domain, frequency-domain and nonlinear) showed no MAP-related changes. It concluded that HRV indices showed significant differences between happiness and sadness emotion states and the findings could help to better understand the inherent differences of cardiovascular time series between different emotion states in clinical practice.