Fungal specific CD154+ T-cells have been described as a biomarker in invasive aspergillosis. The influence of sample storage on the detection of these cells was assessed. Six-hour delay prior to PBMC isolation is associated with an 18% decrease of cell viability and alterations of the cellular composition of the sample. This results in 87% reduction of CD154+A. fumigatus specific cells due to reduced assay sensitivity and increased background values in unstimulated samples. If prompt cell measurement is not feasible, isolated PBMCs can be frozen (at −20°C and −80°C) and processed later with comparable assay reliability (mean value fresh vs. thawing: 0.126, 0.133; Pearson-Coefficient: 0.962).