Intermittent negative pressure (INP) applied to the lower leg induces acute increase in arterial and skin blood flow. The aim of this study was to identify the optimal level of INP to increase blood flow in patients with lower extremity peripheral artery disease (PAD). We investigated the acute effects of different levels of INP in 16 subjects (7 women and 9 men, mean (SD) age 71(8) years) diagnosed with PAD. During application of INP in a pressure chamber sealed below the knee, arterial blood flow was continuously recorded in the dorsalis pedis artery or tibialis posterior artery (ultrasound Doppler), and skin blood flow was continuously recorded at the pulp of the first toe (laser Doppler). Different pressure levels (0, -10, -20, -40, and -60 mmHg) were tested in randomized order. Maximal arterial blood flow relative to baseline (median [25th, 75th percentiles]) was: 0 mmHg; 1.08 (1.02, 1.13), -10 mmHg; 1.11 (1.07, 1.17), -20 mmHg; 1.18 (1.11, 1.32), -40 mmHg; 1.39 (1.27, 1.91) and -60 mmHg; 1.48 (1.37, 1.78). Maximal laser Doppler flux (LDF) relative to baseline was: 0 mmHg; 1.06 (1.02, 1.12), -10 mmHg; 1.08 (1.05, 1.16) -20 mmHg; 1.12 (1.06, 1.27), -40 mmHg; 1.24 (1.14, 1.50) and -60 mmHg; 1.35 (1.10, 1.70). There were significantly higher maximal arterial blood flow and maximal LDF at -40 mmHg compared with -10 mmHg (P = 0.001 and P = 0.025, respectively). There were no significant differences in maximal arterial blood flow and maximal LDF between 0 and -10 mmHg (both P = 1.0), or between -40 and -60 mmHg (both P = 1.0). INP of -40 mmHg was the lowest negative pressure level that increased blood flow.