Aims: Acute mountain sickness (AMS) develops when the body fails to acclimatize to atmospheric changes at altitude. Preascent prediction of susceptibility to AMS would be a useful tool to prevent subsequent harm. Changes to peripheral oxygen saturation (SpO2) on hypoxic exposure have previously been shown to be of poor predictive value. Heart rate variability (HRV) has shown promise in the early prediction of AMS, but its use pre-expedition has not previously been investigated. We aimed to determine whether pre- and intraexpedition HRV assessment could predict susceptibility to AMS at high altitude with better diagnostic accuracy than SpO2. Methods: Forty-four healthy volunteers undertook an expedition in the Nepali Himalaya to >5000 m. SpO2 and HRV parameters were recorded at rest in normoxia and in a normobaric hypoxic chamber before the expedition. On the expedition HRV parameters and SpO2 were collected again at 3841 m. A daily Lake Louise Score was obtained to assess AMS symptomology. Results: Low frequency/high frequency (LF/HF) ratio in normoxia (cutpoint ≤2.28 a.u.) and LF following 15 minutes of exposure to normobaric hypoxia had moderate (area under the curve ≥0.8) diagnostic accuracy. LF/HF ratio in normoxia had the highest sensitivity (85%) and specificity (88%) for predicting AMS on subsequent ascent to altitude. In contrast, pre-expedition SpO2 measurements had poor (area under the curve <0.7) diagnostic accuracy and inferior sensitivity and specificity. Conclusions: Pre-ascent measurement of HRV in normoxia was found to be of better diagnostic accuracy for AMS prediction than all measures of HRV in hypoxia, and better than peripheral oxygen saturation monitoring.