BACKGROUND: Ambulatory children with Spina Bifida (SB) often show a decline in physical activity leading to deconditioning and functional decline. Therefore, assessment and promotion of physical activity is important. Because energy expenditure during activities is higher in these children, the use of existing pediatric equations to predict physical activity energy expenditure (PAEE) may not be valid. AIMS: (1) To evaluate criterion validity of existing predictions converting accelerocounts into PAEE in ambulatory children with SB and (2) to establish new disease-specific equations for PAEE. METHODS: Simultaneous measurements using the Actical, the Actiheart, and indirect calorimetry took place to determine PAEE in 26 ambulatory children with SB. DATA ANALYSIS: Paired T-tests, Intra-class correlations limits of agreement (LoA), and explained variance (R 2 ) were used to analyze validity of the prediction equations using true PAEE as criterion. New equations were derived using regression techniques. RESULTS: While T-tests showed no significant differences for some models, the predictions developed in healthy children showed moderate ICC's and large LoA with true PAEE. The best regression models to predict PAEE were: PAEE = 174.049 + 3.861 × HRAR -60.285 × ambulatory status (R 2 = 0.720) and PAEE = 220.484 + 0.67 × Actical counts -60.717 × ambulatory status (R 2 = 0.681). CONCLUSIONS: Existing equations to predict PAEE are not valid for use in children with SB for the individual evaluation of PAEE. The best regression model was based on HRAR in combination with ambulatory status, followed by a new model for the Actical monitor. A benefit of HRAR is that it does not require the use of expensive accelerometry equipment. Further cross-validation of these models is still needed.