Shift work has been associated with increased risk of cardiovascular disease. This study was designed to determine the hemodynamic effects of 12-hour (12-h) shifts, and changes in blood pressure (BP) and heart rate variability (HRV) during 36 h rest time following 12-h shifts. Fifteen male shift workers with a mean age of 32.9 yr were recruited from a semiconductor factory. Ambulatory BP (AmBP) monitoring was performed for a total of 48 h for each participant. Six workers were monitored for 48 h by Holter electrocardiogram on both the day and night shifts. Paired self-comparison was used to estimate the difference between two hourly measurements of 12-h BP, HR, and HRV using the same timetable intra-individually. We also applied mixed models to estimate the effects of 12-h shifts on the delayed recovery of BP and heart rate (HR) in six workers who completed 96-h AmBP monitoring, including a 48-h night shift-rest period and another day shift period. Results showed that 12-h night shift work gave a persistently elevated systolic and diastolic BP (SBP and DBP) and HR, and decreased HRV compared to 12-h day shift work with the corresponding resting time. In addition, there was delayed SBP and DBP recovery on the first 12-h rest time in night shift workers, which was further demonstrated on the second 12-h rest time after adjustment for possible confounders through mixed models. In conclusion, 12-h night shift work may elevate BP and HR and decrease HRV. It is also associated with delayed BP recovery.