Experimental evidence indicates that exercise performed at different times of the day may affect circadian rhythms and circadian disruption has been linked to breast and prostate cancer. We examined in a population-based case-control study (MCC-Spain) if the time-of-day when physical activity is done affects prostate and breast cancer risk. Lifetime recreational and household physical activity was assessed by in-person interviews. Information on time-of-day of activity (assessed approximately 3 years after the assessment of lifetime physical activity and confounders) was available for 781 breast cancer cases, 865 population female controls, 504 prostate cases and 645 population male controls from 10 Spanish regions, 2008-2013. We estimated odds ratios (ORs) and 95% confidence intervals (95% CI) for different activity timings compared to inactive subjects using unconditional logistic regression adjusting for confounders. Early morning (8-10am) activity was associated with a protective effect compared to no physical activity for both breast (OR = 0.74, 95% CI = 0.48-1.15) and prostate cancer (OR = 0.73, 95% CI = 0.44-1.20); meta-OR for the two cancers combined 0.74 (95%CI = 0.53-1.02). There was no effect observed for breast or prostate cancer for late morning to afternoon activity while a protective effect was also observed for evening activity only for prostate cancer (OR = 0.75, 95% CI = 0.45-1.24). Protective effects of early morning activity were more pronounced for intermediate/evening chronotypes for both cancers. This is the first population-based investigation identifying a differential effect of timing of physical activity on cancer risk with more pronounced effects for morning hour activity. Our results, if confirmed, may improve current physical activity recommendations for cancer prevention.