Background: The impacts of temperature variability on cardiac autonomic function remain unclear. Objective: To explore the short-term associations between daily temperature variability and parameters of heart rate variability (HRV). Methods: This is a repeated-measure study among 78 eligible participants in Shanghai, China. We defined temperature variability as diurnal temperature range (DTR), the standard-deviation of temperature (SDT) and temperature variability (TV). We evaluated 3 frequency-domain HRV parameters (VLF, LF and HF) and 4 time domain parameters (SDNN, SDANN, rMSSD and pNN50). We used linear mixed-effect models to analyze the data after controlling for environmental and individual confounders. Results: Temperature variability was significantly associated with decreased HRV, especially on the concurrent day. The exposure-response relationships were almost inversely linear for most parameters. Every one inter quartile range (IQR) increase of DTR was associated with a decrease of 3.92% for VLF, 6.99% for LF, 5.88% for HF, 3.94% for rMSSD and 1.30% for pNN50. Each IQR increase of SDT was associated with a decline of 6.48% for LF, 5.91% for HF, 4.26% for rMSSD and 1.87% for pNN50. Every IQR increase of SDT was associated with a decrease of 4.39% for VLF, 7.67% for LF, 6.52% for HF, 3.22% for SDNN, 2.98% for SDANN, 4.05% for rMSSD, and 1.41% for pNN50. The decrements in HRV associated with temperature variability were more prominent in females. Conclusion: Temperature variability on the concurrent day could significantly decrease cardiac autonomic function, especially in females.