Purpose: We aimed to establish a core needle biopsy technique to investigate the impact on outcome of irradiation of the microenvironment in individual experimental tumours. Methods: Nude mice bearing FaDu, UT-SCC-5, UT-SCC-14, and UT-SCC-15 tumours (n = 67) were injected with pimonidazole hypoxia and Hoechst 33342 perfusion markers. One core needle biopsy was taken from the central part of the tumour under anaesthesia and the rest of the tumour was excised after marking the position of the needle. Relative hypoxic area (pHF), relative vascular area (RVA), fraction of perfused vessels (PF), and necrotic fraction (NF) were compared in the biopsies and the adjacent whole tumour sections. In a TCD₅₀ (dose to cure 50% of tumours) assay, 223 UT-SCC-5 tumours were irradiated with 30 fractions over 6 weeks either with or without a core biopsy before the start of radiotherapy. Results: The correlations between histological parameters measured in the biopsies and the adjacent tumour sections were dependent on the tumour line. All the four parameters showed weak although significant correlations only in UT-SCC-5. PF was the only parameter which showed a weak but significant correlation in all the four tumour lines. The needle biopsy procedure did not significantly impact on TCD₅₀ after fractionated irradiation in UT-SCC-5: 98 Gy [92; 106] versus 105 Gy [96; 117] (p = 0.12). pHF, RVA, PF, and NF measured in the pre-treatment biopsy did not predict the outcome of fractionated irradiation within the UT-SCC-5 tumour line. Conclusion: A single pre-treatment core needle biopsy may provide valid results for parameters of the tumour micromilieu, however the accuracy is limited by significant intratumoural heterogeneity in the parameters and sampling error. The needle biopsy procedure does not significantly affect local tumour control rates after fractioned irradiation and may therefore be integrated for longitudinal studies in radiobiological experiments. Pre-treatment histological parameters measured in the biopsy did not correlate with the outcome of fractionated irradiation within the UT-SCC-5 tumour line.