PURPOSE: To evaluate the reliability of short-term recordings (five minutes) of heart rate variability (HRV) and the association between HRV and gender. METHODS: HRV time- and frequency-domain parameters were calculated in 44 physically active students (21 males and 23 females) over four consecutive days. A Suunto t6 heart rate monitor was used to obtain inter-beat intervals (IBIs) that were then transferred to Kubios HRV analysis software. The relative reliability [intra-class correlation (ICC)] and absolute reliability, [typical error of measurement (TEM) and typical error of measurement as a percentage (TEM%)] of the HRV parameters were then calculated for day 2 versus day 3 and day 3 versus day 4, with day 1 being a familiarisation day. The following HRV parameters were calculated: (1) time domain: resting heart rate (RHR), R-R intervals (IBI), standard deviation of normal-to-normal intervals (SDNN), root mean square differences of the standard deviation (RMSSD), percentage of beats that changed more than 50 ms from the previous beat (pNN50); and (2) frequency domain: low-frequency normalised units (LFnu), high-frequency normalised units (HFnu), low-frequency to high-frequency ratio in normalised units (LF/HFnu). An analysis of variance (ANOVA) with Tukey post-hoc testing was performed to compare HRV parameters in males and females. Significance was set at p ≤ 0.05. RESULTS: The ICCs for both relationship 1 and 2 indicated primarily good to excellent (> 0.8) relative reliability. The lowest value was found in the LF/HFnu ratio (ICC = 0.36) for males. Absolute reliability was low with TEM% greater than 10% for all HRV parameters, except IBIs. Females demonstrated better relative (higher ICCs) and absolute reliability (lower TEM and TEM% ) compared to males for the frequency domain. The relative and absolute reliability for the time domains were similar except for SDNN where the absolute reliability was higher in males. ANOVA illustrated significant gender differences for the LF/HFnu ratio (41% higher in males, p = 0.003), HFnu (12% higher in females, p = 0.02) and IBI (21% higher in females, p < 0.0001). CONCLUSIONS: Short-term recordings of HRV over three consecutive days demonstrated a high relative reliability. However, a low absolute reliability indicated large day-to-day random variation in HRV, which would make the detection of intervention effects using HRV difficult in individual participants. Females were shown to have a higher parasympathetic modulation of HRV, which may indicate an underlying cardioprotective mechanism in females compared to males.