Background: Time-domain heart rate variability (HRV) indices are commonly used to assess cardiac parasympathetic modulation during short-term recordings. RMSSD is widely recommended for this purpose; however, the sensitivity of NN50 in detecting acute autonomic modulation has not been adequately evaluated. Objectives: To compare the sensitivity of NN50 and RMSSD in detecting acute dietary autonomic modulation using short-term HRV recordings in healthy young adults. Methods: This methodological analysis was conducted on HRV data obtained from 100 healthy young adults who underwent 5-minute resting HRV recordings before and after an acute dietary autonomic stimulus. Time-domain HRV indices NN50 and RMSSD were analysed. Pre–post changes, sex-stratified differences, standardized effect sizes (Cohen’s d and Hedges’ g), and 95% confidence intervals were calculated to assess comparative sensitivity. Results: NN50 showed a statistically significant increase following the intervention (p = 0.015) and demonstrated a larger standardized effect size with confidence intervals excluding zero. In contrast, RMSSD exhibited a non-significant change (p = 0.120) and smaller effect sizes with confidence intervals crossing zero. Sex-stratified analysis revealed greater NN50 responsiveness among females, while RMSSD showed no significant sex-related difference. Conclusion: NN50 is a more sensitive time-domain HRV index than RMSSD for detecting acute dietary autonomic modulation in short-term recordings and should be considered alongside conventional measures in acute autonomic research.