Background: Dynamic preload parameters such as pulse pressure variation (PPV) and stroke volume variation (SVV) have widely been used as accurate predictors for fluid responsiveness in patients under mechanical ventilation. To circumvent the limitation of decreased cyclic change of intrathoracic pressure, we performed an intermittent PEEP challenge test to evaluate whether PPV or SVV can predict fluid responsiveness during one-lung ventilation (OLV). Methods: Forty patients undergoing OLV were analyzed. Baseline hemodynamic variables including PPV and SVV and respiratory variables were recorded after chest opening in lateral position under OLV (T1). Five minutes after application of PEEP 10 cmH2O, the parameters were recorded (T2). Thereafter, PEEP was withdrawn to 0 cmH2O for 5 minutes (T3), and fluid loading was performed with balanced crystalloid solution 6 mL/kg of ideal body weight for 5 minutes. Five minutes after completion of fluid loading, all variables were recorded (T4). The patient was classified as fluid responder if SV increased ≥10% after fluid loading and as non-responder if SV increased <10%. Results: Prediction of fluid responsiveness was evaluated with area under the receiver operating characteristic (ROC) curve (AUC). Change in stroke volume variation (ΔSVV) showed AUC of 0.9 (P < 0.001), 95% CI = 0.82-0.99, sensitivity = 88%, specificity = 82% for discrimination of fluid responsiveness. Change in pulse pressure variation (ΔPPV) showed AUC of 0.88 (P < 0.001), 95% CI = 0.78-0.97, sensitivity = 83%, specificity = 72% in predictability of fluid responsiveness. Cardiac index and stroke volume were well maintained after PEEP challenge in non-responders while they increased in responders. Conclusions: ΔPPV and ΔSVV induced by PEEP challenge are reliable parameters to predict fluid responsiveness as well as very good predictors of fluid unresponsiveness during OLV.