Tumors create an extracellular pH (pHe) barrier and form an isolated microenvironment roughly 0.5 pH units lower than physiological pH, while the intracellular pH (pHi) is maintained within 0.2 pH units of physiological levels. Reduced tumor pHi is associated with apoptosis, and lowered arterial and tissue pH are seen with heavy exercise.PURPOSETo determine the pHi response of MCF‐7 cells subjected to acidic environments. MCF‐7 cells loaded with BCECF, a pH indicator, were used to create a standard curve of fluorescent ratios at six pH levels. MCF‐7 cells were then subjected to pHe levels of 6.75 and 6.25 at 0, 15, 30, 45, 60, 120, 180, and 1,440 minutes of pHe exposure. Data were referenced to MCF‐7 cells at a physiological pHe of 7.25. A treatment effect was seen at pHe 6.25 (p = 0.0002). Compared to the control, cells subjected to pHe 6.25 for 15, 30, 45, and 1,440 minutes had lower pHi levels (p = 0.0464, p = 0.0002, p = 0.0257, and p = 0.0054, respectively). An interaction of pHe and time (p = 0.0483) on pHi was observed. The pHi values were lower at pHe 6.25 than at pHe 6.75 after 30, 180, and 1,440 minutes of exposure (p = 0.0193, p = 0.0200, and p < 0.0001, respectively). In conclusion, MCF‐7 cells subjected to a pHe of 6.75 maintained their pHi, but a different recovery response was seen at pHe 6.25. A decreased tolerance of lower pH levels may provide a mechanism by which heavy exercise would prove beneficial for breast cancer patients.