Purpose: To experimentally validate how temporal modification of the applied dose pattern within a single fraction of radiation therapy affects cell survival. Method and Materials: The linear quadratic (LQ) repair‐time model predicts that, for a single fraction of dose, the degree of cell kill is dependent on the pattern of dose applied over a period of irradiation. Previously, we demonstrated that: (1) maximum cell kill is achieved using a “triangular” temporal dose pattern (delivering the highest doses during the middle of a fraction and the lowest at the beginning and end), and (2) minimum cell kill is achieved with a “V‐shaped” pattern (delivering the lowest doses at the middle of a fraction and the highest at the beginning and end). Furthermore, the model also predicted that cells with low α/β values will have a larger difference in survival based on the applied pattern of dose. Two cells lines with low α/β values (PC‐3, WiDr) and one with a high α/β value (SQ‐20b) were chosen for this study. For each cell line, one group of cells in a six‐well plate received 9 Gy in a triangular dose pattern, and the same dose was delivered to a second plate using a V‐shaped pattern. The delivery time for each dose pattern was 20 min. Cell survival was assessed using a clonogenic assay. Results: For the SQ‐20b cells, irradiation with both dose patterns resulted in only a 4.5% relative difference in cell survival (p>0.25). However, the triangle and V‐shaped patterns resulted in relative cell survival differences of 15.2% and 18.6% for both the PC‐3 (p<0.025) and WiDr (p<0.01) cell lines, respectively. Conclusion: These results verify the assertions of the modeling study in vitro, and imply that the temporal pattern of applied dose is another variable to be considered in treatment planning and delivery.