Diapause survival requires a temperature-sensitive preparatory period

•Diapausing insects are often exposed to warm conditions prior to the onset of winter•Extended fall conditions caused energetic drain and reduced overwintering survival of Choristoneura fumiferana•However, short cool falls also resulted in low survival despite high energy availability•Thus, early diapause onset may be due to an undescribed, temperature sensitive process occurring during diapause initiation that is essential for overwintering survival and post-diapause emergence Diapause is a form of internally-controlled dormancy that allows insects to avoid stressful conditions and periods of low food availability. Eastern spruce budworm (Choristoneura fumiferana Clemens), like many cold-adapted insects, enter diapause well in advance of winter conditions, thus exposing them to elevated temperatures during fall that can deplete energy stores and impact post-diapause survival. We explored the impact of fall conditions on C. fumiferana by manipulating the length of the fall period and exposure temperatures during the diapause initiation phase of second instar larvae in a factorial design. We exposed second instar larvae to four fall temperatures (10, 15, 20, and 25°C) and five exposure times (1, 2, 4, 6, and 10 weeks) prior to standardized diapause conditions. We measured metabolites (glycogen, glycerol, and protein) prior to and during diapause for a subset of individuals. We also measured post-diapause survival by quantifying emergence following diapause conditions for a subset of individuals. We found that long, warm fall conditions depleted glycogen content and lowered post-diapause survival. We also found that short, cool conditions impacted post-diapause survival, although glycogen content remained high. Our results showed that fall conditions have substantial fitness consequences to overwintering insects. Optimal fall conditions struck a balance between exposure time and temperature. Our findings point to a potentially adaptive reason for early diapause onset: that an undescribed, but temperature-sensitive process is occurring in C. fumiferana larvae during the diapause initiation period that is essential for overwintering survival and successful post-diapause emergence.