Expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) is important in predicting a response to targeted therapies in breast cancer. Immunohistochemical assays to determine hormone receptor (HR) and HER2 status must therefore be accurate and reproducible. Tissue fixation has been shown to play a crucial role in determining consistency in quality. Although guidelines impose upper limits for the fixation period, the data on which these limits are based are scant. This study aimed to prospectively examine the effect of fixation of >72 hours on these assays. In 101 invasive breast cancer samples, HR and HER2 status was compared between tumor blocks undergoing a short fixation period with those undergoing a period of prolonged fixation (PF). Discordances were classified as an incremental change between categories of (i) a single order of magnitude, that is a difference in the status of low positive (Allred score 3) compared with positive (Allred score 4 to 8) or negative (Allred score 0 or 2) and vice versa for HRs and a difference in HER2 status of equivocal compared with negative or positive and vice versa or (ii) greater than a single order of magnitude, that is a difference in the status of positive compared with negative or vice versa. The median fixation time for the short fixation group was 13 hours and 18 minutes (mean, 13 h 17 min; range, 10 h 33 min to 17 h 45 min) and for the PF group was 79 hours 22 minutes (mean, 79 h 35 min; range, 73 h 33 min to 102 h 30 min). Eight cases showed discordances, all of which were of a single order of magnitude including 1 for ER, 5 for PR, and 2 for HER2. In 6 of these, a higher score was seen in the PF group. In conclusion, fixation for limited periods beyond 72 hours does not result in a reduction in assay sensitivity in the determination of ER, PR, or HER2 immunohistochemical status.