Fecal volatile organic compounds (VOCs) are increasingly considered to be potential noninvasive, diagnostic biomarkers for various gastrointestinal diseases. Knowledge of the influence of sampling conditions on VOC outcomes is limited. We aimed to evaluate the effects of sampling conditions on fecal VOC profiles and to assess under which conditions an optimal diagnostic accuracy in the discrimination between pediatric inflammatory bowel disease (IBD) and controls could be obtained. Fecal samples from de novo treatment-naïve pediatric IBD patients and healthy controls (HC) were used to assess the effects of sampling conditions compared to the standard operating procedure (reference standard), defined as 500 mg of sample mass diluted with 10 mL tap water, using field asymmetric ion mobility spectrometry (FAIMS). A total of 17 IBD (15 CD (Crohn's disease) and 2 UC (ulcerative colitis)) and 25 HC were included. IBD and HC could be discriminated with high accuracy (accuracy = 0.93, AUC = 0.99, p < 0.0001). A smaller fecal sample mass resulted in a decreased diagnostic accuracy (300 mg accuracy = 0.77, AUC = 0.69, p = 0.02; 100 mg accuracy = 0.70, AUC = 0.74, p = 0.003). A loss of diagnostic accuracy was seen toward increased numbers of thaw-freeze cycles (one cycle, accuracy = 0.61, AUC = 0.80, p = 0.0004; two cycles, accuracy = 0.64, AUC = 0.56, p = 0.753; and three cycles, accuracy = 0.57, AUC = 0.50, p = 0.5101) and when samples were kept at room temperature for 180 min prior to analysis (accuracy = 0.60, AUC = 0.51, p = 0.46). Diagnostic accuracy of VOC profiles was not significantly influenced by storage duration differences of 20 months. The application of a 500 mg sample mass analyzed after one thaw-freeze cycle showed the best discriminative accuracy for the differentiation of IBD and HC. VOC profiles and diagnostic accuracy were significantly affected by sampling conditions, underlining the need for the implementation of standardized protocols in fecal VOC analysis.